Retrocyclins: antiviral and antimicrobial peptides

ABSTRACT

Retrocyclin peptides are small antimicrobial agents with potent activity against bacteria and viruses. The peptides are nonhemolytic, and exhibit minimal in vitro cytotoxicity. A pharmaceutical composition comprising retrocyclin as an active agent is administered therapeutically to a patient suffering from a bacterial and/or viral infection, or to an individual facing exposure to a bacterial and/or viral infection, especially one caused by the HIV-1 retrovirus or other sexually-transmitted pathogens.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under grantnumber AI22839 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

[0002] Natural polycationic antimicrobial peptides have been found inmany different species of animals and insects and shown to have broadantimicrobial activity. In mammals, these antimicrobial peptides arerepresented by two families, the defensins and the cathelicidins. Nearlyall of these peptides have membrane affinity, and can permeate andpermeabilize bacterial membranes, resulting in injury, lysis, and/ordeath to the microbes. In particular, the human peptides known asdefensins are produced by mammalian and avian leukocytes (e.g.neutrophils, some macrophages) and epithelial cells.

[0003] Three defensin subfamilies exist in vertebrates: alpha-defensins,beta-defensins, and circular (theta) minidefensins. All derive from anancestral gene that existed before reptiles and birds diverged, containsix cysteines, and have largely beta-sheet structures that arestabilized by three intramolecular disulfide bonds. RTD-1, a thetaminidefensin, was recently detected in bone marrow from the rhesusmonkey, Macacca mulatta. It had 18 residues and was circular, havingbeen formed by the fusion of two truncated alpha-defensin precursors(“demidefensins”) each of which contributed 3 cysteines to the maturepeptide. The cellular machinery responsible for processing theseprecursors remains operational in human leukocytes.

[0004] Alpha-defensins are largely beta sheet peptides that contain29-35 amino acid residues, including 6 cysteines that form threeintramolecular disulfide bonds. Because of the nature of the cysteinepairings, the molecules are effectively macrocyclic. Four of thesea-defensins, HNP 1-4, occur primarily in human neutrophils. HD-5 & 6 arefound in Paneth cells, specialized cells of the small intestine'scrypts. Human α-defensin genes contain three exons and two introns andare clustered on chromosome 8p23. They encode preprodefensins thatcontain˜100 residues which encode a signal peptide, polyanionic propieceand the C-terminal defensin domain. Mature defensins are processed bysequential proteolysis.

[0005] Beta defensins are generally larger than α-defensins (35-40residues) and may also be more ancient, since they occur in birds aswell as mammals. Beta defensins are expressed in many different types ofepithelial cells, and in some glands. In some cases, expression isconstitutive; in others, it is inducible. Several β-defensin genes arelocated on 8 p23, adjacent to the α-defensin genes—consistent with theircommon evolutionary ancestry. The disulfide pairing motif of betadefensins differs from that of α-defensins, however α and β-defensinshave generally similar shapes.

[0006] The three-dimensional structure of many defensins comprises acomplexly folded amphiphillic beta-sheet, with the polar face formed byits arginines and by the N- and C-terminal residues playing an importantrole in defining microbicidal potency and the antimicrobial spectrum.The antimicrobial effects of defensins are derived from their ability topermeabilize cell membranes and interact with viral envelopes, therebyexposing contents of the microorganism to the environment or abrogatingviral infectivity. (See Gudmundsson et al. (1999) J Immunol Methods232(1 -2):45-54.) Antimicrobial peptides are reviewed by Hancock andLehrer (1998) Trends in Biotechnology 16:82.

[0007] In general, the antiviral activities of antimicrobial peptideshave not been extensively investigated. Although studies have reportedthat antimicrobial peptides, such as human neutrophil-derived defensins(α-defensins), are directly virucidal against herpes simplex virus(HSV), and adenovirus strains, only a few reports deal with anti-HIV-1activity. T22 and T140, analogs of polyphemusins (peptides fromhorseshoe crabs), are active in inhibiting HIV-1; replication throughbinding to the chemokine receptor CXCR4. However, these peptides onlyinhibit the T cell-tropic (T-tropic; X4) strains that utilize CXCR4 as acoreceptor for entry and they are ineffective against strains thatutilize CCR5 for entry (macrophage (M)-tropic “R5” viruses). Sincesexual transmission is largely attributed to R5 infection, the potentialof T22 and T140 as topical vaginal or rectal microbicides is limited.

[0008] One study indicated that protegrins (porcine-derived peptides)can inactivate HIV-1 virions. Another study showed that indolicidin, a13 amino acid peptide isolated from bovine neutrophils, was reproduciblyvirucidal against HIV-1 only at very high concentrations (333 μg/ml) ofpeptide. While the anti-HIV-1 activity of human α-defensins has not beenreported, certain structural and functional similarities exist betweenthe loop motifs of α-defensins and peptides derived from HIV-1 gp41 thatmay be required for viral fusion and infectivity.

[0009] Vaginal and rectal subepithelial stromal tissues are denselypopulated with dendritic cells (DC), macrophages and T-cells thatexpress both CD4 and the HIV-1 coreceptors, CXCR4 and CCR5. Mechanismswhereby HIV-1 journeys across the mucosal epithelia are not clear, butmay directly involve the epithelial cells. Once the virus reaches thelamina propria, it can either directly infect macrophages or T-cells oradhere to or infect DC whose traffic to the regional lymph nodes conveysthem into sites of vigorous viral replication. A recent report suggeststhat binding of HIV-1 to DC is mediated by the C-type lectin DC-SIGN,independent of CD4 or chemokine receptors. Thus, mucosal factors whichmodulate steps in this process could affect the probability oftransmission of HIV-1 infection.

[0010] There is a clinical need for novel antiviral and antimicrobialagents that have low toxicity against mammalian cells. The presentinvention addresses this need.

[0011] Relevant literature

[0012] Defensins are reviewed by Lehrer et al. (1992) Ann. Rev. Immunol.11:105-128. Other endogenous antimicrobials are reviewed in Schonwetteret al. (1995) Science 267:1645-1648; Schroder (1999)Cell Mol Life Sci.56:32-46 (1999); and Harwig et al. (1994) FEBS Lett 342:281 -285.

[0013] Specific defensins are described in Tang et al. (1999) Science286:498-502; Zimmermann et al. (1995) Biochemistry 34:13663-13671; Liuet al. (1997) Genomics 43:316-320; and Palfree & Shen (1994)GenBankU10267; Polley et al. GenBankAF238378disclose the sequence ofHomo sapiens chromosome 8p23 clone SCb-561 b 17.

[0014] Retrovirus infection and antiretroviral therapy are discussed inWilson et al. (1995) J. Infect. Dis. 172:88-96; Wong et al. Science278:1291-1295; and Yang et al.(1999) J. Virol. 73, 4582-4589.

SUMMARY OF THE INVENTION

[0015] Methods and compositions are provided for the use of retrocyclinpeptides. Retrocyclin peptides are small antimicrobial agents withpotent activity against viruses, e.g. enveloped viruses such asretroviruses; and bacteria. These circular peptides are nonhemolytic andgenerally exhibit little or no in vitro cytotoxicity. Retrocyclins areequally effective against growing and stationary phase bacteria, andthey retain activity against some bacteria in physiological, and highsalt concentrations. Studies indicate that retrocyclins are also capableof conferring immunity to human CD4⁺ cells against infection by HIV-1 invitro. In addition, other circular mini-defensins also find use asanti-viral agents, particularly against human retroviruses.

[0016] A pharmaceutical composition comprising retrocyclin or othercircular mini-defensins as an active agent is administered to a patientsuffering from a viral infection. Alternatively, a pharmaceuticalcomposition comprising retrocyclin or other circular mini-defensins oris administered as a protective agent to a normal individual facingpotential exposure to HIV viruses or pathogenic microbes. Retrocyclin isalso effective at killing a variety of microbial organisms, in vivo andin vitro. Retrocyclin may be administered alone, or in combination withother bacteriocidal agents, e.g. antibiotics and/or other antiviralagents, and antiviral agents as a cocktail of effective peptides, etc.Retrocyclin-mediated killing is also useful for modeling and screeningnovel antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1. Circular minidefensins reduce HIV-1 infection of H9 cells.HIV-I strain IIIB (MOI =10⁻²) was incubated with 2.5×10⁵ H9 cells in thepresence or absence of 20 μg/ml RTD-1, RTD-2 or RTD-3. p24 antigenrelease was monitored by ELISA on days 3, 6, and 9. Assay sensitivity=10 pg/ml.

[0018]FIG. 2. Sequence comparison of human and rhesus demidefensins. Thetranslated sequences of rhesus demidefensin-1 mRNA and human retrocyclinmRNA are shown. Solid circles () indicate a stop codon in thecorresponding cDNA. Vertical bars connect identical residues, and +signs connect similar residues. Residues represented in matureretrocyclin and RTD-molecules are boxed. The demidefensin-1 sequence(GenBank, AF184156) was derived from the monkey mRNA (not shown).

[0019]FIG. 3. Structural characterization of retrocyclin. (A) CDspectrum demonstrating the similarity in structure between retrocyclinand RTD1, both at 0.5 mg/ml in a 1:1mixture of trifluoroethanol inphosphate buffered saline at pH 7.4. (B) shows a hypothetical model ofretrocyclin made by templating its sequence on the backbone of a similarpeptide from porcine neutrophils, Protegrin-1 (PDB accession code:1PG1). (D) is a cartoon version of (B), wherein arginines are black,cysteines are grey and the other residues are identified by singleletter code. (C) is a similar cartoon of rhesus RTD-1, indicating thesimilarity in structure with retrocyclin.

[0020]FIG. 4. Effect of salt on antibacterial activity of circularminidefensins. Human retrocyclin and monkey RTD-1 were tested againstour standard lab stains: E. coli ML-35p, P. aeruginosa MR 3007, L.monocytogenes EGD, and S. aureus 930918. The bars show MIC values ± SEMvalues that resulted from 3-6 radial diffusion assays per organism andassay condition.

[0021]FIG. 5. Anti-HIV-1 activity of retrocyclin. Two strains of HIV-1and two types of human target cells were used. The IIIB strain is T-celltropic (X4) and utilizes the CXCR4 co-receptor for entry; the JR-CSFstrain is M-tropic (R5) and uses CCR5 for entry. PBMC signifiesCD4⁺-selected peripheral blood mononuclear cells. Results indicate p24antigen concentration in pg/ml, as determined by quantitative ELISAassay at Day 9 timepoint. (A) Two concentrations of retrocyclin (2μg/ml, 20 μg/ml), 20 μg/ml of the Rhesus circular defensin “RTD-1”, and20 μg/ml of a horseshoe crab-derived peptide “T140”, reported to onlyprevent X4 infections, were tested in antiviral assays of against strainIIIB in H9 cells (n=2-6 per peptide; error bars indicate SEM). (B) Toconfirm our results with primary human cells, similar assays wereperformed utilizing IIIB virus and CD4⁺PBMC or (C) JR-CSF virus andCD4⁺PBMC. Peptides were not cytotoxic at indicated concentrations,measured by trypan blue exclusion. Average of duplicate experiments arereported for studies with PBMCs. Assay sensitivity =10 pg/ml.

[0022]FIG. 6. Retrocyclin can inhibit HIV-1 spread when administered upto 24 hrs post-infection. Primary CD4⁺PBMC were incubated with HIV-IIIBfor 3 hours in the absence (“control”, “t0”, “t3”, and “t24”) orpresence (“t0only”) of 20 μg/ml retrocyclin. Cells were transferred tofresh R10-50 media that was either supplemented immediately with 20μg/ml retrocyclin (“t0”), or 3 or 24 hrs after transfer (“t3”and “t24”,respectively). “Control” and “tΦ only” were not supplemented aftertransfer. p24 antigen was measured by ELISA as previously described.

[0023]FIG. 7. Mature retrocyclin, but not premature forms, inhibit HIV-1replication. H9 cells were incubated with HIV-IIIB (MOI=10⁻²) for 3hours in the absence or presence of 20 μg/ml retrocyclin in threeflavors: linear and reduced; linear and oxidized disulfide bonds; andthe mature form (cyclic and oxidized). Assay sensitivity is 10 pg/ml.

[0024]FIG. 8. Cytotoxicity of antimicrobial peptides against H9 cells.Retrocyclin, RTD-1 and PG-1 (a porcine-derived peptide with anti-HIV-1activity) were tested for cytotoxicity using an MTT assay for cellproliferation. Note that the EC₅₀ of Retrocyclin and RTD-1 were >100μg/ml, concentrations well above their antiviral concentration.

[0025]FIGS. 9A and 9B are graphs depicting the activity of retrocyclincongeners against HIV-1 strains.

[0026]FIGS. 10A and 10B are schematics depicting the structure ofretrocyclin.

[0027] FIGS. 11A-C compares the antiretroviral activity of retrocyclinand RC-101 (20 μg/ml), by showing the p24 titers from day 9 CD4⁺PBMC(peripheral blood mononuclear cells) infected with HIV-1 strains IIIB orJR-CSF at the indicated MOI. RC-101 and retrocyclin were similarlyeffective in inhibiting HIV-1 replication at low MOI (A) and higher MOI(B).

[0028]FIG. 12. Adding retrocyclin directly with HIV-1-IIIB does notreduce infection of H9 cells. Retrocyclin (2-200 μg/ml) was incubatedwith HIV-IIIB (MOI=10⁻²) diluted in R10 media prior to infecting H9cells. p24 antigen release was measured by ELISA. Limit of detection =10pg/ml.

[0029]FIG. 13. Retrocyclin and RC-101 inhibited the formation of HIVproviral DNA. Retrocyclin and RC-101 inhibited the formation of DNA fromboth early events (total HIV DNA) and later events (full-length HIV DNA)of reverse transcription. Data are an average of 2 experiments, exceptfor RC-101 (1 experiment). “HI virus” is a heat-inactivated viruscontrol for background levels of viral DNA.

[0030]FIG. 14. Inactivation of HSV-1 and HSV-2 by retrocyclin andRC-101. Retrocyclin (left panel) and RC-101 (right panel) at theindicated concentrations were incubated with herpes simplex virus, type1 (HSV-1) or HSV-2 for 2 hrs and then added to ME-180 cell monolayers.Cells were incubated at 37° C. for 72 hrs, and cytotoxicity was measuredwith an MTT kit.

[0031]FIG. 15 depicts sequences of human, ape and monkey retrocyclins.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0032] Novel compositions and methods are provided for the use ofretrocyclins and retrocyclin analogs as therapeutic and/or prophylacticagents. The peptides are effective at killing a variety of microbialorganisms by direct microbicidal activity, and protect against viralinfection by a virus by preventing viral uptake and/or blocking an earlystep in viral replication. Retrocyclin(s) are administered alone or incombination with other active agents to a patient suffering from aninfection in a dose and for a period of time sufficient to reduce thepatient population of pathogenic microbes or viruses. Alternatively, apharmaceutical composition comprising retrocyclin or other circularmini-defensins or is administered as a protective agent to a normalindividual facing potential exposure to HIV viruses or pathogenicmicrobes. In addition, other circular mini-defensins, including RTD-1,RTD-2 and RTD-3 and variants of retrocyclin find use as anti-viralagents.

[0033] Specific treatments of interest include, without limitation:using retrocyclin (e.g., RC-101) or a retrocyclin analog to prevent ortreat infection, for example by an enveloped virus, including envelopedretroviruses, more specifically by HIV-1, HIV-2 and related retrovirusesthat cause Acquired Immunodeficiency Syndrome (AIDS); aerosoladministration to the lungs of patients with cystic fibrosis to combatinfection or forestall the emergence of resistance to other inhaledantibiotics; instillation into the urinary bladder of patients withindwelling catheters to prevent infection; application to the skin ofpatients with serious bums; opthalmic instillation, directly or inophthalmic solutions, to treat or prevent infection; intravaginalapplication to treat bacterial vaginosis and/or prevent sexuallytransmitted disease such as HIV infection. The retrocyclins also mayfind use in the treatment of plant-pathogenic pseudomonads, inagricultural applications designed to prevent disease in and spoilage offood crops. The retrocyclins may be administered alone or in conjunctionwith other antiviral therapy.

[0034] The peptide form of retrocyclins provides a basis for furthertherapeutic development, by modification of the polypeptide structure toyield modified forms having altered biological and chemical properties.The native or modified forms are formulated in a physiologicallyacceptable carrier for therapeutic uses, or are otherwise used as anantimicrobial agent.

Retrocyclin Compositions

[0035] For use in the subject methods, a naturally occurring orsynthetic retrocyclin may be used. As used herein, retrocyclins arecyclic polypeptides comprising the amino acid sequence: X₁ X₂ X₃ X₄ X₅X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈

[0036] wherein X1 and X18 are linked through a peptide bond,

[0037] disulfide crosslinks are formed between at least one of: X₃ andX₁₆; X₅ and X₁₄, and X₇ and X₁₂, usually between at least two of suchpairs, and preferably between the three pairs of amino acids, with theproviso that when such a crosslink is present, the crosslinked aminoacids are both cysteines;

[0038] at least about three of amino acids X₁ to X₁₈ are arginine orlysine, and the number of arginine or lysine residues may be four ormore, five or more, or six or more. Preferred residues for arginine orlysine are X₄, X₉, X₈, X₁₃, and X₁₈;

[0039] X₂, X₅, X₁₁, X₁₅ are preferably aliphatic amino acids, e.g.isoleucine, leucine, valine, phenylalanine, and alanine;

[0040] X₁, X₈, X₁₀ and X₁₇ are preferably glycine or alanine, usuallyglycine.

[0041] Retrocyclins are octadecapeptides that contain two linkednonapeptides that may be identical or different. A consensus nonapeptidehas the sequence shown below, where the bolded and underlined residuesare invariant among the primate sequences identified herein.Substitutions found in the nonapeptide regions of other circularminidefensin precursors are shown below the consensus nonapeptide.Residue No 1 3 5 7 9 Consensus nonapeptide RC I C GRGI C Variant   LRLRV Variant     T  F Variant     V Variant       R

[0042] From the consensus peptide and these variants, one can generateunique nonapeptide sequences (herein termed n1, n2 . . . etc. ). Thus,n1 could be linked to itself or any of the other nonapeptides (n1:n1,n1:n2, n1:n3 . . . etc.), to generate unique octadecapeptides. Tocontinue the process, n2 could be linked to itself or to any othernonapeptide except n1, to generate additional unique octadecapeptides,and so forth.

[0043] Two naturally occurring human nonapeptide sequences areRCICGRGIC; and RCICGRRIC. The set of nonapeptides derived from thesesequences and variants (which are also provided in the sequence listingas SEQ ID NO: 19-64; and 94-119) is as follows: NP^(#) 1 2 3 4 5 6 7 8 9mod'n* NP^(#) 1 2 3 4 5 6 7 8 9 modification* 1 R C I C G R G I C — 24 RC I C G L G F C L6, F8 2 R C L C G R G I C L3 25 R C I C G L G V C L6,V8 3 R C I C R R G I C R5 26 R C L C R L G I C L3, R5, L6 4 R C I C T RG I C T5 27 R C L C R R G V C L3, R5, V8 5 R C I C V R G I C V5 28 R C LC R R G F C L3, R5, F8 6 R C I C G L G I C L6 29 R C L C T L G I C L3,T5, L6 7 R C I C G R G V C V8 30 R C L C G R G V C L3, T5, V8 8 R C I CG R G F C F8 31 R C L C T R G F C L3, T5, F8 9 R C L C R R G V C L3, R532 R C L C V L G I C L3, V5, L6 10 R C L C T R G I C L3, T5 33 R C L C VR G V C L3, V5, V8 11 R C L C V R G I C L3, V5 34 R C I C G R G I C L3,V5, F8 12 R C L C G L G V C L3, L6 35 R C I C R L G V C R5, L6, V8 13 RC L C G R G V C L3, V8 36 R C I C R L G F C R5, L6, F8 14 R C L C G R GF C L3, F8 37 R C I C T L G V C T5, L6, V8 15 R C I C R R G V C R5, V838 R C I C T L G F C T5, L6, F8 16 R C I C R R G F C R5, F8 39 R C I C VL G V C V5, L6, V8 17 R C I C T R G V C T5, V8 40 R C I C V L G F C V5,L6, F8 18 R C I C T R G F C T5, F8 41 R C L C G L G V C L3, R5, L6, V819 R C I C T L G I C T5, L6 42 R C L C G L G I C L3, R5, L6, F8 20 R C IC V L G F C V5, L6 43 R C L C T L G V C L3, T5, L6, V8 21 R C I C R L GI C R5, L6 44 R C L C T L G I C L3, T5, L6, F8 22 R C I C V R G V C V5,V8 45 R C L C V L G V C L3, V5, L6, V8 23 R C I C G R G F C V5, F8 46 RC L C V L G I C L3, V5, L6, F8 47 R C I C G R R I C — 70 R C I C G L R FC L6, F8 48 R C L C G R R I C L3 71 R C I C G L R V C L6, V8 49 R C I CR R R I C R5 72 R C L C R L R I C L3, R5, L6 50 R C I C T R R I C T5 73R C L C R R R V C L3, R5, V8 51 R C I C V R R I C V5 74 R C L C R R R FC L3, R5, F8 52 R C I C G L R I C L6 75 R C L C T L R I C L3, T5, L6 53R C I C G R R V C V8 76 R C L C G R R V C L3, T5, V8 54 R C I C G R R FC F8 77 R C L C T R R F C L3, T5, F8 55 R C L C R R R V C L3, R5 78 R CL C V L R I C L3, V5, L6 56 R C L C T R R I C L3, T5 79 R C L C V R R VC L3, V5, V8 57 R C L C V R R I C L3, V5 80 R C I C G R R I C L3, V5, F858 R C L C G L R V C L3, L6 81 R C I C R L R V C R5, L6, V8 59 R C L C GR R V C L3, V8 82 R C I C R L R F C R5, L6, F8 60 R C L C G R R F C L3,F8 83 R C I C T L R V C T5, L6, V8 61 R C I C R R R V C R5, V8 84 R C IC T L R F C T5, L6, F8 62 R C I C R R R F C R5, F8 85 R C I C V L R V CV5, L6, V8 63 R C I C T R R V C T5, V8 86 R C I C V L R F C V5, L6, F864 R C I C T R R F C T5, F8 87 R C L C G L R V C L3, R5, L6, V8 65 R C IC T L R I C T5, L6 88 R C L C G L R I C L3, R5, L6, F8 66 R C I C V L RF C V5, L6 89 R C L C T L R V C L3, T5, L6, V8 67 R C I C R L R I C R5,L6 90 R C L C T L R I C L3, T5, L6, F8 68 R C I C V R R V C V5, V8 91 RC L C V L R V C L3, V5, L6, V8 69 R C I C G R R F C V5, F8 92 R C L C VL R I C L3, V5, L6, F8

[0044] Retrocyclins of interest include cyclic peptides derived from thepeptide sequence set forth in SEQ ID NO. 12, in particular a circularhomodimer comprising a dimer of the amino acid sequence SEQ ID NO: 12,aa 48-56. This retrocyclin has the structure (SEQ ID NO: 1):G   I   C   R   C   I   C   G   R   G   I   C   R   C   I   C   G   R X₁ X₂  X₃  X₄  X₅  X₈  X₇  X₈  X₉  X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈

[0045] Wherein X₁ and X₁₈ are joined by a peptide bond, X₂ and X₁₁; X₄and X₉, and X₁₃ and X₁₈ are disulfide bonded.

[0046] Another retrocyclin of interest is the synthetic analog (SEQ IDNO: 2) G   I   C   R   C   I   C   G   KG   I   C   R   C   I   C   G   R X₁  X₂  X₃  X₄  X₅  X₆  X₇  X₈  X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈

[0047] wherein X₁ and X₁₈ are joined by a peptide bond, X₂ and X₁₁; X₄and X₉, and X₁₃ and X₁₈ are disulfide bonded. Other synthetic analogs,or congeners, of retrocyclin are set forth in SEQ ID NO: 3-SEQ ID NO:10.

[0048] The sequence of the retrocyclin polypeptides may be altered invarious ways known in the art to generate targeted changes in sequence.The polypeptide will usually be substantially similar to the sequencesprovided herein, i.e. will differ by one amino acid, and may differ bytwo amino acids. The sequence changes may be substitutions, insertionsor deletions.

[0049] The protein may be joined to a wide variety of otheroligopeptides or proteins for a variety purposes. By providing forexpression of the subject peptides, various post-translationalmodifications may be achieved. For example, by employing the appropriatecoding sequences, one may provide famesylation or prenylation. In thissituation, the peptide will be bound to a lipid group at a terminus, soas to be able to be bound to a lipid membrane, such as a liposome.

[0050] Modifications of interest that do not alter primary sequenceinclude chemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

[0051] Also included in the subject invention are polypeptides that havebeen modified using ordinary molecular biological techniques andsynthetic chemistry so as to improve their resistance to proteolyticdegradation or to optimize solubility properties or to render them moresuitable as a therapeutic agent. Analogs of such polypeptides includethose containing residues other than naturally occurring L-amino acids,e.g. D-amino acids or non-naturally occurring synthetic amino acids.

[0052] The subject peptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

[0053] If desired, various groups may be introduced into the peptideduring synthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

[0054] The polypeptides may also be isolated and purified in accordancewith conventional methods of recombinant synthesis. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. For the most part, the compositionswhich are used will comprise at least 20% by weight of the desiredproduct, more usually at least about 75% by weight, preferably at leastabout 95% by weight, and for therapeutic purposes, usually at leastabout 99.5% by weight, in relation to contaminants related to the methodof preparation of the product and its purification. Usually, thepercentages will be based upon total protein. Genetic sequences encodingdemi-defensins are provided herein, e.g. SEQ ID NO: 4, 7 and 9.

[0055] In one embodiment of the invention, the antimicrobial peptideconsists essentially of a polypeptide sequence set forth in any one ofSEQ ID NO: 1-SEQ ID NO: 10. By “consisting essentially of” in thecontext of a polypeptide described herein, it is meant that thepolypeptide is composed of the sequence set forth in the seqlist, whichsequence may be flanked by one or more amino acid or other residues thatdo not materially affect the basic characteristic(s) of the polypeptide.

[0056] For some purposes of the invention, for example in the treatmentand/or prevention of HIV infection, the active agent may be any one ofthe circular minidefensins, e.g. human retrocyclins, RTD-1, RTD-2 andRTD-3. Cyclic minidefensins resemble protegrins, antimicrobial , sheetpeptides. RTD-1 is derived from Macacca mulatta, and is a heterodimercontaining tandem nonapeptide elements derived from the mature peptidesset forth in SEQ ID NO: 15 and SEQ ID NO: 17. RTD-2 is a homodimercontaining, in tandem, two identical nonapeptide elements derived fromthe mature peptide set forth in SEQ ID NO: 17. RTD-3 is a homodimercontaining, in tandem, two identical nonapeptide elements derived fromthe mature peptide set forth in SEQ ID NO: 15.

[0057] All three RTD's are circular molecules with 18 residues and threeintramolecular disulfide bonds. Each RTD is formed by in vivo processingthat trims and splices two precursor peptides (“demidefensins”), each ofwhich contributes nine residues (including 3 cysteines) to the maturecyclic peptide. The 18RTD-1 residues derive from two differentdemidefensin precursors, RTD-2 and -3 have tandem 9 residue repeatsderived from a single demidefensin precursor.

Retrocyclin Coding Sequences

[0058] The invention includes nucleic acids having a sequence set forthin SEQ ID NO: 11, 120 or 122; nucleic acids that hybridize understringent conditions, particularly conditions of high stringency, to thesequence set forth in SEQ ID NO: SEQ ID NO: 11, 120 or 122; genescorresponding to the provided nucleic acids; sequences encodingretrocyclins; and fragments and derivatives thereof. Other nucleic acidcompositions contemplated by and within the scope of the presentinvention will be readily apparent to one of ordinary skill in the artwhen provided with the disclosure here. Genetic sequences of particularinterest include primate sequences, e.g. human, chimpanzee, bonobo,orangutan, gorilla, etc.

[0059] Retrocyclin coding sequences can be generated by methods known inthe art, e.g. by in vitro synthesis, recombinant methods, etc. toprovide a coding sequence to corresponds to a linear retrocyclinpolypeptide that could serve as an intermediate in the production of thecyclic retrocyclin molecule. Using the known genetic code, one canproduce a suitable coding sequence. For example, the circularpolypeptide of retrocyclin (SEQ ID NO: 1) is encoded by the sequence(SEQ ID NO: 18) AGG TGC ATT TGC GGA AGA GGAATT TGC AGG TGC ATT TGC GGAAGA GGA ATT TGC, but since the peptide is circular, it is somewhatarbitrary which codon is selected to be first, allowing this to be basedon other criteria, e.g. relative efficiency in purification orcyclization of the predicted product. The polypeptide set forth in SEQID NO: 2 is encoded by a similar sequence, wherein one of the argininecodons (AGA) is substituted with a lysine codon (AAA or AAG).

[0060] The nucleic acids of the invention include nucleic acids havingsequence similarity or sequence identity to SEQ ID NO: SEQ ID NO: 11,18, 120 or 122. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 10 XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1XSSC. Sequence identity can bedetermined by hybridization under stringent conditions, for example, at50° C. or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate).Hybridization methods and conditions are well known in the art, see,e.g., U.S. Pat. No. 5,707,829. Nucleic acids that are substantiallyidentical to the provided nucleic acid sequence, e.g. allelic variants,genetically altered versions of the gene, etc., bind to SEQ ID NO: SEQID NO: 11, 18, 120 or 122 under stringent hybridization conditions. Byusing probes, particularly labeled probes of DNA sequences, one canisolate homologous or related genes. The source of homologous genes canbe any species, e.g. primate species, particularly human; rodents, suchas rats and mice; canines, felines, bovines, ovines, equines, fish,yeast, nematodes, etc.

[0061] In one embodiment, hybridization is performed using at least 18contiguous nucleotides (nt) of SEQ ID NO: 1 and SEQ ID NO: 18, or a DNAencoding the polypeptide of SEQ ID NO: 1-10, 19-64, or 74-119. Such aprobe will preferentially hybridize with a nucleic acid comprising thecomplementary sequence, allowing the identification and retrieval of thenucleic acids that uniquely hybridize to the selected probe. Probes ofmore than 18 nt can be used, e.g., probes of from about 18 nt to about25, 50, 100 or 250 nt, but 18 nt usually represents sufficient sequencefor unique identification.

[0062] Nucleic acids of the invention also include naturally occurringvariants of the nucleotide sequences (e.g., degenerate variants, allelicvariants, etc.). Variants of the nucleic acids of the invention areidentified by hybridization of putative variants with nucleotidesequences disclosed herein, preferably by hybridization under stringentconditions. For example, by using appropriate wash conditions, variantsof the nucleic acids of the invention can be identified where theallelic variant exhibits at most about 25-30% base pair (bp) mismatchesrelative to the selected nucleic acid probe. In general, allelicvariants contain 15-25% bp mismatches, and can contain as little as even5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.

[0063] The invention also encompasses homologs corresponding to thenucleic acids of SEQ ID NO: 5, where the source of homologous genes canbe any mammalian species, e.g., primate species, particularly human;rodents, such as rats; canines, felines, bovines, ovines, equines, fish,yeast, nematodes, etc. Between mammalian species, e.g., human and mouse,homologs generally have substantial sequence similarity, e.g., at least75% sequence identity, usually at least 90%, more usually at least 95%between nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 contiguous nt long, moreusually at least about 30 nt long, and may extend to the completesequence that is being compared. Algorithms for sequence analysis areknown in the art, such as gapped BLAST, described in Altschul et al.Nucl. Acids Res. (1997) 25:3389-3402.

[0064] The subject nucleic acids can be cDNAs or genomic DNAs, as wellas fragments thereof, particularly fragments that encode a biologicallyactive polypeptide and/or are useful in the methods disclosed herein.The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding apolypeptide of the invention.

[0065] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It can further include the 3′ and 5′untranslated regions found in the mature mRNA. It can further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA can be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ and 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue, stage-specific, or disease-state specificexpression.

[0066] The nucleic acid compositions of the subject invention can encodeall or a part of the subject polypeptides. Double or single strandedfragments can be obtained from the DNA sequence by chemicallysynthesizing oligonucleotides in accordance with conventional methods,by restriction enzyme digestion, by PCR amplification, etc. Isolatednucleic acids and nucleic acid fragments of the invention comprise atleast about 18, about 50, about 100, to about 200 contiguous nt selectedfrom the nucleic acid sequence as shown in SEQ ID NO: SEQ ID NO: 11, 120or 122. For the most part, fragments will be of at least 18 nt, usuallyat least 25 nt, and up to at least about 50 contiguous nt in length ormore.

[0067] Probes specific to the nucleic acid of the invention can begenerated using the nucleic acid sequence disclosed in SEQ ID NO: 11, ora DNA encoding the polypeptide of SEQ ID NO: 1-10. The probes arepreferably at least about 18 nt, 25nt or more of the correspondingcontiguous sequence. The probes can be synthesized chemically or can begenerated from longer nucleic acids using restriction enzymes. Theprobes can be labeled, for example, with a radioactive, biotinylated, orfluorescent tag. Preferably, probes are designed based upon anidentifying sequence of one of the provided sequences. More preferably,probes are designed based on a contiguous sequence of one of the subjectnucleic acids that remain unmasked following application of a maskingprogram for masking low complexity (e.g., BLASTX) to the sequence, i.e.,one would select an unmasked region, as indicated by the nucleic acidsoutside the poly-n stretches of the masked sequence produced by themasking program.

[0068] The nucleic acids of the invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which: it: is not normally associated on a naturally occurringchromosome.

[0069] Retrocyclin encoding nucleic acids can be provided as a linearmolecule or within a circular molecule, and can be provided withinautonomously replicating molecules (vectors) or within molecules withoutreplication sequences. Expression of the nucleic acids can be regulatedby their own or by other regulatory sequences known in the art. Thenucleic acids of the invention can be introduced into suitable hostcells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

[0070] Expression vectors may be used to introduce a retrocyclin codingsequence into a cell. Such vectors generally have convenient restrictionsites located near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

[0071] The gene or retrocyclin peptide may be introduced into tissues orhost cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992)Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992) Nature 356:152-154), where gold microprojectiles arecoated with the stresscopin or DNA, then bombarded into skin cells.

Methods of Use

[0072] Formulations of retrocyclins are administered to a host sufferingfrom an ongoing bacterial or viral infection or who faces exposure to abacterial or viral infection. Antiviral compositions may also utilizeother circular mini-defensins, e.g. RC-101, RTD-1, -2, and -3, alone orin combination with retrocyclin. Administration may be topical,localized or systemic, depending on the specific microorganism.Generally the dosage will be sufficient to decrease the microbial orviral population by at least about 50%, usually by at least 1 log, andmay be by 2 or more logs. The compounds of the present invention areadministered at a dosage that reduces the pathogen population whileminimizing any side-effects. It is contemplated that the compositionwill be obtained and used under the guidance of a physician for in vivouse. Retrocyclins are particularly useful for killing Listeriamonocytogenes and Escherichia coli, and for preventing infection bycertain viruses, particularly enveloped retroviruses, e.g. envelopedretroviruses such as HIV-1, HIV-2, FIV, and the like.

[0073] Retrocylins are also useful for in vitro formulations to killmicrobes, particularly where one does not wish to introduce quantitiesof conventional antibiotics. For example, retrocyclins may be added toanimal and/or human food preparations, or to blood products intended fortransfusion to reduce the risk of consequent bacterial or viralinfection. This may be of particular interest since a common route ofinfection of E. coli and L. monocytogenes is the gastrointestinal tract.Retrocyclins may be included as an additive for in vitro cultures ofcells, to prevent the overgrowth of microbes in tissue culture.

[0074] The susceptibility of a particular microbe or virus to killing orinhibition by retrocyclins may be determined by in vitro testing, asdetailed in the experimental section. Typically a culture of the microbeis combined with retrocyclins at varying concentrations for a period oftime sufficient to allow the protein to act, usually ranging from aboutone hour to one day. The viable microbes are then counted, and the levelof killing determined. Two stage radial diffusion assay is a convenientalternative to determining the MIC or minimum inhibitory concentrationof an antimicrobial agent.

[0075] Viral pathogens of interest include retroviral pathogens, e.g.HIV-1; HIV-2, HTLV, FIV, SIV, etc. Microbes of interest, but not limitedto the following, include: Citrobacter sp.; Enterobacter sp.;Escherichia sp., e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteussp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium;Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa;Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Y enterocolitica;Franciscella sp.; Pasturella sp.; Vibrio sp., e.g. V. cholerae, V.parahemolyticus; Campylobacter sp., e.g. C. jejuni; Haemophilus sp.,e.g. H. influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, B.bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp., e.g. N.gonorrhoeae, N. meningitidis, etc. Other bacteria of interest includeLegionella sp., e.g. L. pneumophila; Listeria sp., e.g. L.monocytogenes; Staphylococcus sp., e.g. S. aureusMycoplasma sp., e.g. M.hominis, M. pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M.leprae; Treponema sp., e.g. T pallidum; Borrelia sp., e.g. B.burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R. rickettsii, R.typhi; Chlamydia sp., e.g. C. trachomatis, C. pneumoniae, C.psittaci;Helicobacter sp. e.g. H. pylon, etc.

[0076] Various methods for administration may be employed. For theprevention of HIV infection, administration to mucosal surfaces is ofparticular interest, e.g. vaginal, rectal, etc. The polypeptideformulation may be given orally, or may be injected intravascularly,subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder,topically, etc. For example, methods of administration by inhalation arewell-known in the art. The dosage of the therapeutic formulation willvary widely, depending on the specific retrocyclin or demi-defensin tobe administered, the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the host, and the like. The initial dose may be larger, followed bysmaller maintenance doses. The dose may be administered as infrequentlyas weekly or biweekly, or fractionated into smaller doses andadministered once or several times daily, semi-weekly, etc. to maintainan effective dosage level. In many cases, oral administration willrequire a higher dose than if administered intravenously. The amidebonds, as well as the amino and carboxy termini, may be modified forgreater stability on oral administration.

Formulations

[0077] The compounds of this invention can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, the compounds of the present invention can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, lotions, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, vaginal, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intratracheal, etc.,administration. The retrocyclins may be systemic after administration ormay be localized by the use of an implant or other formulation that actsto retain the active dose at the site of implantation.

[0078] The compounds of the present invention can be administered alone,in combination with each other, or they can be used in combination withother known compounds (e.g., perforin, anti-inflammatory agents,antibiotics, etc.) In pharmaceutical dosage forms, the compounds may beadministered in the form of their pharmaceutically acceptable salts. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

[0079] For oral preparations, the compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0080] The compounds can be formulated into preparations for injectionsby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

[0081] The compounds can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0082] The compounds can be used as lotions, for example to preventinfection of burns, by formulation with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

[0083] Furthermore, the compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

[0084] Unit dosage forms for oral, vaginal or rectal administration suchas syrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore compounds of the present invention. Similarly, unit dosage formsfor injection or intravenous administration may comprise the compound ofthe present invention in a composition as a solution in sterile water,normal saline or another pharmaceutically acceptable carrier.

[0085] Implants for sustained release formulations are well-known in theart. Implants are formulated as microspheres, slabs, etc. withbiodegradable or non-biodegradable polymers. For example, polymers oflactic acid and/or glycolic acid form an erodible polymer that iswell-tolerated by the host. The implant containing retrocyclins isplaced in proximity to the site of infection, so that the localconcentration of active agent is increased relative to the rest of thebody.

[0086] The term “unit dosage form”, as used herein, refers tophysicallydiscrete units suitable as unitary:dosages for human andanimal subjects, each unit containing a predetermined quantity of:compounds of the present invention calculated in an amount sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with the compound in the host.

[0087] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0088] Typical dosages for systemic administration range from 0.1 μg to100 milligrams per kg weight of subject per administration. A typicaldosage may be one tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient. The time-releaseeffect may be obtained by capsule materials that dissolve at differentpH values, by capsules that release slowly by osmotic pressure, or byany other known means of controlled release.

[0089] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

[0090] The use of liposomes as a delivery vehicle is one method ofinterest. The liposomes fuse with the cells of the target site anddeliver the contents of the lumen intracellularly. The liposomes aremaintained in contact with the cells for sufficient time for fusion,using various means to maintain contact, such as isolation, bindingagents, and the like. In one aspect of the invention, liposomes aredesigned to be aerosolized for pulmonary administration. Liposomes maybe prepared with purified proteins or peptides that mediate fusion ofmembranes, such as Sendai virus or influenza virus, etc. The lipids maybe any useful combination of known liposome forming lipids, includingcationic or zwitterionic lipids, such as phosphatidylcholine. Theremaining lipid will be normally be neutral or acidic lipids, such ascholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[0091] For preparing the liposomes, the procedure described by Kato etal. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids andlumen composition containing peptides are combined in an appropriateaqueous medium, conveniently a saline medium where the total solids willbe in the range of about 1-10 weight percent.. After intense agitationfor short periods of time, from about 5-60 sec., the tube is placed in awarm water bath, from about 25-40° C. and this cycle repeated from about5-10 times. The composition is then sonicated for a convenient period oftime, generally from about 1-10 sec. and may be further agitated byvortexing. The volume is then expanded by adding aqueous medium,generally increasing the volume by about from 1-2 fold, followed byshaking and cooling. This method allows for the incorporation into thelumen of high molecular weight molecules.

Formulations with Other Active Agents

[0092] For use in the subject methods, retrocyclins may be formulatedwith other pharmaceutically active agents, particularly otherantimicrobial agents. Other agents of interest include a wide variety ofantibiotics, as known in the art. Classes of antibiotics includepenicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin,carbenicillin, nafcillin, ampicillin, etc.; penicillins in combinationwith β-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin,cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides;tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides;quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim;vancomycin; etc.

[0093] Cytokines may also be included in a retrocyclin formulation, e.g.interferon γ, tumor necrosis factor α, interleukin 12, etc.

[0094] Antiviral agents, e.g. acyclovir, gancyclovir, etc., and othercircular mini-defensins (theta defensins) may also be included inretrocyclin formulations.

Experimental

[0095] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

[0096] Sexual and mother-to-neonate (vertical) transmission throughmucosal surfaces have been the most common routes of HIV-1 spreadthroughout the world. Although much attention has been focused onvaccine development for HIV-1, progress has been slow and there is anurgent need to find alternative approaches to prevent infections causedby HIV-1. Self-applied prophylactic agents to prevent mucosal,particularly vaginal or rectal, transmission of HIV-1 have the advantageof empowering vulnerable receptive partners to take effective measuresfor their own protection. In a search for novel compounds active againstHIV-1, it was discovered that certain antimicrobial peptides, thecircular minidefensins from the rhesus macaque, could inhibit HIV-1replication. This prompted an investigation as to whether humans producecircular minidefensins. Although there is no evidence that theseproteins are produced in humans, clearly some primate ancestors oncemade retrocyclin, because it continues to exist in contemporary humansas an expressed pseudogene.

[0097] After discovering an mRNA molecule in human bone marrow that washighly homologous to rhesus circular minidefensins (88.9% identity atthe nucleotide level), solid phase peptide synthesis was used to createthe peptide (“retrocyclin”) whose sequence it encoded. Retrocyclinbelongs to the θ defensin subfamily (also referred to as cyclicminidefensins). The antimicrobial properties of retrocyclin resemblethose of rhesus θ-defensins. However, retrocyclin is highly effective inpreventing the infection of CD4⁺ cells by X4 and R5 strains of HIV-1 invitro.

EXAMPLE 1 Circular Minidefensins can Block HIV-1 Replication

[0098] It is shown herein that retrocyclin, a circular minidefensin, ispotently active against both X4 and R5 strains of HIV-1. The initialdescriptions of circular minidefensins came from studies of Macacamulatta, the rhesus macaque monkey. The first such peptide, RTD-1, wascalled a rhesus theta defensin (RTD), which are also referred to as“cyclic minidefensins”. The peptides encoded by the mRNA precursors maybe referred to as “demidefensins”.

[0099] RTD-2 and RTD-3, which was isolated from the bone marrow ofrhesus monkeys, are circular, 18 amino acid peptides that containedthree intramolecular disulfide bonds. They are similar to RTD-1,thecircular (θ) defensin previously described by Tang et a. However,whereas the 18 residues of RTD-1 represent spliced 9 amino acidfragments derived from two different minidefensin precursors, RTD-2 and-3 comprise tandem 9 residue repeats derived from a single RTD-1precursor. Thus, circular minidefensins are processed by a novelpost-translational system that can generates a degree of effectormolecule diversity without requiring commensurate genome expansion.

[0100] Retrocyclin and the other circular minidefensins we prepared weresynthesized, folded, circularized and purified essentially. Theantiviral activities of RTD-1, RTD-2 and RTD-3 are shown in FIG. 1. Forthese studies, the X4 HIV-1 strain IIIB was utilized.

[0101] Immortalized CD4⁺H9 cells, which are permissive for infectionwith this strain, were maintained in RPMI supplemented with 10%heat-inactivated fetal calf serum (FCS), 10 mM HEPES,2 mM glutamine, 100U of penicillin/ml, and 10 μg of streptomycin/ml (R10 media). Cells(2.5×10⁵/100 μl) were incubated with virus (multiplicity of infection(MOI)=10⁻²) in the presence or absence of 20 μg/ml RTD-1, RTD-2 or RTD-3for 3 hrs at 37° C./5% CO₂. The cells were washed in R10 media, seededin 48-well tissue culture plates in 1 ml R10 media, and incubated at 37°C./5% CO₂ for 9 days. Aliquots of cell supernatant were removed at thespecified time points and analyzed by a sensitive ELISA (DuPont NEN)that quantitates p24 antigen of HIV-1. The three circular rhesusminidefensins were similarly active, inhibiting HIV-1 by 100-1000 foldby 9 days post-inoculation (note the logarithmic scale).

EXAMPLE 2 Identification and Structural Characterization of Retrocyclin.

[0102] To search for human circular minidefensins, two primers wereprepared based on the monkey minidefensin cDNA sequences (GenBank AF184156, 184157,184158). When PCR was performed on Marathon-Ready humanbone marrow cDNA (Clontech, Palo Alto, Calif.), a 264 bp amplifiedproduct was recovered. To obtain its 3′ and 5′ side sequences,Marathon-Ready human bone marrow cDNA was amplified using a 3′-RACE kit(Gibco BRL, Gaithersburg, Md.) and 5′-RACE kit from Boehringer Mannheim(Indianapolis, Ind.).

[0103] At the nucleotide level, this product (retrocyclin) was 89%identical to the demidefensin precursors of rhesus RTD-1 (calledprecursors 1 a and 1 b). FIG. 2 shows the peptide sequences ofdemidefensin 1 and preproretrocyclin. Residues incorporated into themature circular minidefensins are boxed and all stop codons arerepresented by solid circles. Although a stop codon within the humantranscript's signal sequence should abort translation, the otherwisehigh conservation of rhesus and human mRNA's suggested that humans mayhave acquired this mutation relatively recently in primate evolution.

[0104] Three orangutan retrocyclin genes have been sequences. One ofthese climes has the silencing stop codon in the signal sequence andtherefore resembles human retrocyclin. The other two orangutan genesappear to be functional, i.e. when translated they would producedemi-defensins, the precursors of cyclic minidefensins.

[0105] Human leukocytes were examined for the presence of retrocyclin orsimilar peptides, but, as expected from the presence of the signalsequence's stop codon, none was found: Thus synthetic retrocyclinrepresents the circular minidefensin that would have formed: a) if thesignal sequence mutation were absent, and b) if the precursor underwenthomologous pairing so that its boxed residues (see FIG. 2) formed bothhalves of the circular molecule.

[0106] In phylogenetic studies of the retrocyclin demidefensin gene, thepremature stop codon in the signal sequence was found to be present infour anthropoid species (humans, gorillas, chimpanzees, pygmychimpanzees) and not present in the genes of a fifth (orangutangs). Thedemidefensin gene also appears intact (i.e., no premature stop codon) inthe two catarrhine (Old World Monkey) species examined to date, Macacamulatta and Macaca nemestrina. These findings suggest that nativeretrocyclin peptides were last produced by a primate ancestor of humansand other anthropoids that lived between 6 and 15 million years ago(mya). This is between the time that orangutang and human lineagediverged (15 mya) and before the divergence of the chimpanzee and humanlineages (6 mya). These ongoing studies of primate phylogeny may yieldsequence information about additional cyclic minidefensins whose nativecounterparts are extinct.

[0107] Retrocyclin synthesis

[0108] Peptides were synthesized at a 0.25 mmol scale with aPerkin-Elmer ABI431A Synthesizer, using pre-derivatized polyethyleneglycol polystyrene arginine resin (PerSeptive Biosystems, Framingham,Mass.), FastMoc™ chemistry, and double coupling for all residues. Thecrude peptide was reduced under nitrogen, for 15 hours at 50° C. withexcess dithiothreitol in 6 M guanidine.HCl, 0.2 M Tris.HCl and 0.2 mMEDTA (pH 8.2). The reaction was stopped with glacial acetic acid (finalconcentration, 5%) and the reduced peptide was stored under nitrogenuntil purified by RP-HPLC. After this step, the peptide appearedhomogeneous and its mass (1942.5, by MALDI-TOF MS) agreed well with itstheoretical mass. The reduced peptide (0.1 mg/ml) was oxidized, cyclizedand purified essentially as described by Tang et al., supra. TheMALDI-TOF MS mass of retrocyclin (1918.5 Da) agreed well with itsexpected mass. CD spectra were obtained at 25° C. from an AVIV 62DSspectropolarimeter (AVIV, Lakewood, N.J.).

[0109] RTD-1 and retrocyclin have very similar CD spectra, with largelyβ-sheet structures stabilized by disulfide linkages and connected byturns (FIG. 3A). Antimicrobial peptides with similar spectra includetachyplesins, protegrins, and circularized defensins. FIG. 3B, abackbone ribbon model of retrocyclin, was made by templating itssequence on the structure of protegrin PG-1 and cyclizing it. Theresulting structure was annealed by molecular dynamics and energyminimized. FIG. 3D is a cartoon version of FIG. 3B, designed primarilyto show the placement of the cysteine and arginine molecules. FIG. 3C isa similar cartoon of rhesus RTD-1.

[0110] Retrocyclin is a selectively salt-insensitive antibacterialpeptide

[0111] The effects of NaCl on the antimicrobial activity of retrocyclinand RTD-1, from two-stage radial diffusion assays, are compared in FIG.4. The peptides showed very similar behavior. Under low salt conditions,both peptides were highly effective (minimal inhibitory concentration(MIC)<3 μg/ml) against all four test organisms: Pseudomonas aeruginosa,Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.Their strong activity against E. coli and L. monocytogenes persisted inphysiological (100 mM) NaCl, and even hypersalinity (175 mM NaCl) wasonly modestly inhibitory. In contrast, neither peptide was effective(MIC>50 μg/ml) against S. aureus or P. aeruginosa in physiological orhigh salt concentrations. Retrocyclin's activity is likely to bepreserved in the ionic concentration of the vaginal mucosa.

[0112] Retrocyclin potently inhibits HIV-1 replication of R5 and X4viruses

[0113] The antiretroviral properties of retrocyclin are shown in FIG. 5.Either HIV-1-permissive H9 cells were used as targets, or primary CD4⁺lymphocytes from HIV-1-seronegative donors generated from freshlypurified peripheral blood mononuclear cells (PBMC) stimulated with aCD3-CD8 bispecific monoclonal antibody. After approximately 7 days, when98% of these cells co-expressed CD3 and CD4, they were infected withHIV-1 with or without retrocyclin or other test peptide. These cellswere maintained in RPMI containing 10% FCS supplemented with 2 mMglutamine, 100 U of penicillin/ml, 10 μg of streptomycin/ml, and 50 U ofinterleukin 2/ml (R10-50 media).

[0114] Retrocyclin (10-20 μg/ml) afforded complete suppression of viralreplication to CD4⁺-selected PBMC challenged with two different strainsof HIV-1: IIIB (an X4 strain) and JR-CSF (an R5 strain), or H9 human Tcells challenged with IIIB. Note that the concentration of p24 antigenis presented on a log-scale and that the rhesus circular minidefensins,RTD-1 (FIG. 5 and FIG. 1) and RTD-2 and RTD-3 were protective to alesser extent than retrocyclin. Additionally, the antiretroviralactivities of T140 (20 μg/ml; FIG. 5) and T22, analogs of polyphemusinsfrom horseshoe crabs that were previously shown to protect against X4,but not R5, infections, were confirmed in the present study.Microbicides, such as retrocyclin, that target both X4 and R5 viruses bemore effective than agents that preferentially inhibit viruses of asingle tropism.

[0115] Examining the effect of adding retrocyclin at various times pre-and post-HIV-1 infection

[0116] To determine if retrocyclin is effective against HIV-1 when addedpost-infection, we either: 1) added retrocyclin at the time of HIV-1infection, then washed away the peptide, or 2) added retrocyclin atvarious times post-infection. Primary CD4⁺PBMC were incubated withHIV-IIIB (FIG. 6) or HIV-JR-CSF for 3 hours in the presence or absenceof 20 μg/ml retrocyclin. The cells were subsequently washed in media,and incubated an additional 9 days. Retrocyclin (20 μg/ml) was addedback to some of the cultures at time points specified in FIG. 6 andinfection was monitored by p24 ELISA as previously described. Althoughretrocyclin was most active when administered at the time of infection,and when present in culture throughout the 9 day incubation, retrocyclinadministered as late as 24 hours after initial infection still reducedthe p24 concentration by nearly 1000-fold.

[0117] Cyclization and oxidation are necessary for retrocyclin'santiviral activity

[0118] Mature retrocyclin was prepared by a three-step process. Its twointermediate forms, as well as the final retrocyclin product were testedin our standard assay of HIV-1 infectivity: p24 ELISA of HIV-IIIBinfection of H9 cells (FIG. 7). Intermediate 1 (open triangles) is thelinearized retrocyclin octadecapeptide with 6 reduced cysteine thiolgroups. Intermediate 2 (closed triangles) is a noncyclic b-hairpinoctadecapeptide with 3 intramolecular cystine disulfide bonds.Retrocyclin (open squares), is a cyclized octadecapeptide with 3disulfide bonds. Note that only the “mature” form of retrocyclin wasantiviral, compared to control (no retrocyclin, open circles). Unlikeretrocyclin, the linearized octadecapeptide was highly cytotoxic, asmeasured by trypan blue exclusion, and treated cells did not survivepast 6 days.

[0119] Retrocyclin is not cytotoxic

[0120] Cytotoxicity determinations were made with a Cell ProliferationKit from Boehringer Mannheim used according to the manufacturer'sinstructions. The procedure measures the reduction of the yellowish MTTmolecule (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) to a dark blue formazan. Retrocyclin exhibited little to nocytotoxicity against H9 cells (FIG. 8) and ME-180 cervical carcinomacells at 100 μg/ml, a concentration that is far higher than theconcentrations required for complete protection against HIV-1 infection(10 μg/ml). Additionally, neither 20 μg/ml retrocyclin nor RC-101 werecytotoxic to HIV-1-infected H9 cells and CD4⁺PBMC as measured bytrypan-blue exclusion (Table 1). Retrocyclin was not hemolytic for humanerythrocytes. TABLE 1 Cytotoxicity of 20 μg/ml peptide against H9 cellsand CD4⁺ PBMC (peripheral blood mononuclear cells) as measured by Trypanblue exclusion. Cells; virus no peptide* Retrocyclin CD4⁺ PBMC; no virus1.07 0.98 H9; IIIB 0.78 1.20 CD4⁺ PBMC; IIIB 1.71 1.68 CD4⁺ PBMC; JR-CSF0.90 1.58

[0121] Construction and characterization of retrocyclin congeners

[0122] To date, we have constructed over a dozen congeners ofretrocyclin, “RC-101”, “RC-102”, “RC-103”, etc. and have used them tocommence a structure-activity analysis of the retrocyclin's antiviraland antimicrobial effects. These peptides, whose sequences are shown inTable 2, were synthesized, oxidized, cyclized, and purified as describedabove for retrocyclin. RC-101 was prepared because retrocyclin, acircular peptide without free N-terminal or side-chain amine groups, isnot well suited for fluorescent-conjugation. RC-101 is identical insequence to retrocyclin (RC-100) except for the presence of an Arg₉→Lys₉substitution. This modification preserves the net cationic charge of thepeptide and provides an available epsilon-amino group in lysine's sidechain. Importantly, RC-101 was as active as retrocyclin in protectingcells from infection by HIV-1 (FIG. 11), indicating that substitutionsin the primary sequence of retrocyclins can be made without losinganti-retroviral activity. The labeling of RC-101 with amine-reactiveprobes will be described in a later section.

[0123] Five additional analogues (RC 110-114) have been synthesized,cyclized and purified. RC-110 (Inverso-enantioretrocyclin), a cyclicpeptide composed exclusively of D amino acids, has a sequence that isidentical to retrocyclin, but with its residues placed in reverse order.

[0124] The ability of RC100 and several analogues described in Table 2to protect cells from infection by X4 (HIV-IIIB) and R5 (JR-CSF) strainsof HIV-1 is shown in FIG. 9. The structure of retrocyclin itself isshown in FIG. 10, with its residues numbered to correspond to Table 2.

[0125] The p24 assay results shown in FIG. 9 are on a logarithmic scale.A horizontal reference line that passes through 100 on the ordinatescale corresponds to 1 pg/ml of p24 antigen. Results from 3 experiments(each performed with PBMC from a different donor) are shown. Retrocyclinwas uniformly protective against both strains of HIV-1 in all of theexperiments. Most of the mono-tyrosine substituted amino acid congenersof retrocyclin (RC102-RC-103, RC-105, RC-106, and RC-108) were eitherinactive or only modestly active in inhibiting HIV-1 infection by StrainIIIB. In contrast, RC-102, RC-103 and RC-104 showed considerable abilityto protect cells from infection by the JR-CSF strain (R5).

[0126] These results allow some hypotheses about the mechanism of actionof retrocyclins to be formulated. Because RC-112 (enantioRetrocyclin)was relatively ineffective, chiral interactions between retrocyclin andone or more receptors on the cell and/or virus surface are likely toparticipate in the protective mechanism. Since certain analogues(RC-102, RC-103 and perhaps RC104) manifested substantial activityagainst the R5 strain but were relatively ineffective against the X4strain, the mechanisms whereby retrocyclin inhibits these strains arenot identical. The lack of efficacy of RC-106, RC-107 and RC-108 (eachcontaining a tyrosine for arginine replacemene) suggests that ionicinteractions involving the positively charged arginine residues inposition 4,9, and 13 of retrocyclin (see the model in FIG. 10) withoppositely charged groups (e.g., phosphate) on the surface of the targetcell or HIV-1 virion also participate in the process. In preliminarysurface plasmon resonance (SPR) experiments, we have observed thatretrocyclin s with high affinity to certain sphingolipids (e.g.,galactosylceramide) that are present in cell-surface rafts, and havebeen implicated in the cellular uptake of HIV-1 virions. TABLE 2 Primaryamino acid sequence of selected retrocyclin congeners. Name Avg. MWAmino acid sequence SEQ ID NO: Peptide (or comment) (Da) 1 RC-100*Retrocyclin 1918.4 GICRCICGRGICRCICGR 2 RC-101 R₉K-Retrocyclin 1890.4GICRCICGKGICRCICGR 3 RC-102 I₆Y-Retrocyclin 1968.5 GICRCYCGRGICRCICGR 4RC-103 I₁₅Y-Retrocyclin 1968.5 GICRCICGRGICRCYCGR 5 RC-104I₂Y-Retrocyclin 1968.5 GYCRCICGRGICRCICGR 6 RC-105 I₁₁Y-Retrocyclin1968.5 GICRCICGRGYCRCICGR 7 RC-106 R₄Y-Retrocyclin 1925.4GICYCICGRGICRCICGR 8 RC-107 R₉Y-Retrocyclin 1925.4 GICICICGYGICRCICGR 9RC-108 R₁₃Y-Retrocyclin 1925.4 GICICICGRGICYCICGR 10 RC-109**GICICICGRGICRCICGY 19 RC-110 Inverso-enantio-Retrocyclin 1918.4RGCICRCIGRGCTCRCIG (ALL D) 20 RC-111 Inverso-retrocyclin 1918.4RGCICRCIGRGCICRCIG 21 RC-112 enantio-retrocyclin 1918.4GICRCICGRGICRCICGR (all D) 22 RC-113 enantio-RC-101 1890.4GICRCICGKGICRCICGR (all D) 23 RC-114 RC-101/103 1940.4GICRCICGKGICRCYCGR hybrid

[0127] Retrocyclin does not directly inactivate HIV-1.

[0128] To determine if retrocyclin directly inactivated HIV-1 virions,HIV-IIIB (MOI 10⁻²) was incubated with 2 μg/ml, 20 μg/ml, or 200 μg/mlretrocyclinfor 30 min at room temperature in R10 media. The mixture wasdiluted 190-fold in R10 media, to dilute retrocyclin below its effectiveantiviral concentrations (no significant antiviral activity at <2 μg/ml;n=5), and used to infect 5×10⁵ H9 CD4⁺ cells. Viral replication wasmeasured by collecting supernatant for 9 days at 3 day intervals toquantify HIV-1 p24 antigen by ELISA (FIG. 12). HIV titer was not reducedwith the highest concentration (200 μg/ml) of retrocyclin, demonstratingthat retrocyclin does not target the virion directly. In this respect,the actions of retrocyclin are different from the direct inactivation ofherpes simplex virus previously observed with human and rabbitα-defensins.

[0129] Retrocyclin binds to T1 cells

[0130] Since retrocyclin does not directly inactivate HIV-1 virions, theability of retrocyclin to interact with a cellular target wasdetermined, using RC-101, a Arg₉→Lys₉ congener of retrocyclin thatretained the antiretroviral activity of the parent molecule. RC-101 wasconjugated to the amine-reactive fluorescent dye, BODIPY-FL (MolecularProbes), according to the manufacturer's protocol. The conjugate(RC-101_(BODIPY-FL)) was purified by reverse-phase HPLC and resuspendedin 0.01% acetic acid at up to 240 μg/ml. RC-101_(BODIPY-FL) (20 μg/ml)was incubated with 2.5×10⁵ CD4⁺-selected PBMC cells for 15 min at roomtemperature, washed once in fresh R10-50 media. Specimens were imaged ona Leica TCS-SP Confocal Microscope (Heidelberg, Germany) equipped withan argon laser for excitation of BODIPY-FL and phycoerythrin (PE).Images were collected with Leica Confocal Software. RC-101_(BODIPY-FL)bound to the cell membrane, mostly in patches. Patching(“microaggregation”) has been reported to occur with hormone-occupiedepidermal growth factor receptors (95), and “rafts” are involved insignaling through the confinement of chemokine receptors to discreteregions of the cell membrane. RC-101_(BODIPY-FL) colocalizes withphycoerythrin (PE)-labeled monoclonal antibodies directed against CXCR4,CCR5 and CD4, but does not with PE-labeled isotype control antibodies.Thus, retrocyclin aggregates in the same “rafts” as the receptor andcoreceptors for HIV-1. In addition, RC-101_(BODIPY-FL) aggregated inpatches where CD4, CXCR4 and CCR5 levels were weak or absent

[0131] A flow cytometry experiment was performed to examine binding ofBODIPY- labeled RC-101. T1 cells were incubated for 1 hr at 37° C.±20μg/ml RC-101_(BODIPY-FL), washed with R10 media, and fixed in 2%paraformaldehyde/PBS. Cells were analyzed by fluorescence-activated cellsorting (FACS) on a Becton-Dickinson FACScan. Live cells (10⁴ events)were gated and analyzed by CellQuest. Two peaks were present, which mayrepresent non-specific and specific cellular binding

[0132] Retrocyclin inhibits HIV replication at an early step (reversetranscription or before)

[0133] To determine whether retrocyclin blocked the formation ofproviral DNA in HIV-JR-CSF-inoculated CD4⁺-selected PBMC, quantitativereal time PCR was performed. This method is more sensitive thanmeasuring p24 release and can detect infection even when p24values maybe affected by virus carried over from the original inoculum.CD4⁺-selected PBMC (10⁶ cells)were incubated in 250 μl at 37° C./5% CO₂for 3 hr with either HIV-1 strain JR-CSF (MOI=0.1), heat-inactivatedvirus (background control), JR-CSF +20 μg/ml retrocyclin or JR-CSF +20μg RC-101. Cells were washed and resuspended in 1 ml R10-50 media, andincubated for an additional 9 hours. Following incubation, cells werepelleted at 300× g, removed of overlying supernatant, and stored at −80°C. until analyzed by real time PCR. Retrocyclin and RC-101 inhibited theformation of HIV-1 proviral DNA (FIG. 13), indicating that retrocyclinacts early, either to inhibit reverse transcription or preceding events.

[0134] Some retrocyclins are slightly active against herpes simplexvirus (HSV)

[0135] To determine if the activity of retrocyclin against HIV-1 wasspecific or representative of a more global antiviral effect, it wastested its ability to prevent HSV infection in vitro. A quantitativemicroplate assay was used to screen retrocyclin andretrocyclin-congeners for their ability to inactivate HSV type 1 (HSV-1)and HSV-2. The assay utilized small amounts of peptide andsimultaneously evaluated for peptide-induced cytotoxicity. In brief,final peptide concentrations of 2-50 μg/ml were incubated with virusstocks for 2 hrs and added directly to ME-180 human cervical carcinomacells. Cultures were incubated at 37° C. for 72 hrs and cytotoxicity wasmeasured using a 3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell proliferation kit (Boehringer-Mannheim, Germany).Calculations to assess antiviral activity and compute “percentprotection” are delineated in (90). Retrocyclin afforded modestprotection against HSV-2, but not HSV-1 (FIG. 14). In contrast, RC-102and RC-103 were less antiviral than retrocyclin. However, RC-101 wasmodestly protective against HSV-1 and nearly completely protectiveagainst HSV-2. The substitution of Arg₉→Lys₉ produced a retrocyclincongener that retained activity against HIV-1, bacteria and fungi, andwas more active against HSV.

EXAMPLE 3 Sequences of Retrocyclins

[0136] We have obtained samples of DNA from the 22 primate specieslisted below in the Table. The first column shows the phylogeneticgroup, and has the following key: E. Great apes/humans; C: Old WorldMonkeys; B: New World Monkeys, D. Lesser Apes; A. Column 3, showing“Genetic distance” from Homo sapiens, is expressed in %. These valuescome from study of gamma globin DNA (Page, SL., Chiu C, and Goodman, M.Molecular phylogeny of Old World Monkeys (Cercopithecidae) as inferredfrom gamma-globin DNA sequences. Molecular Phylogenetics and Evolution.1999. 13:348-359.) Column four shows the sources of DNA samples: 1, theCoriell Institute; and 2, the “Frozen Zoo” collection of the San DiegoZoo. We have performed RT-PCR on all of these samples with primers basedon the sequence of the human retrocyclin gene. “Pending” means that thestudies are still undergoing confirmation.

[0137] All of the great apes (Group E) have a retrocyclin gene orpseudogene. In the gorilla and chimpanzee the gene was inactivated bythe identical signal sequence stop codon mutation found in the humanretrocyclin pseudogene. A Sumatran orangutan DNA sample obtained fromthe Coriell Institute contained two retrocyclin genes. One genecontained the signal sequence mutation and the other appeared intact.Additional orangutan DNA samples have been obtained from the “FrozenZoo” (described below) and are being characterized. None of the 6 NewWorld monkeys (Group B) or 5 the prosimian species (Group A) had aretrocyclin gene. TABLE 3 Genetic DNA Retrocyclin Retrocyclin GroupSpecies Distance Source Gene Gene Status E Homo sapiens 0.0 1 Presentinactivate E Pan troglodytes (chimpanzee) 1.6 1 Present inactivate E Panpaniscus (Bonobo) 1.7 1 Present inactivate E Gorilla gorilla (Lowlandgorilla) 1.9 1 Present inactivate E Pongo pygmaeus pyg. (Borneoorangutan) 3.1 2 pending pending E Pongo pygmaeus abeli (Sumatraorangutan) 3.1 1,2 Present intact/inactivate D Hylobates syndactylus(siamang) 4.2 2 Present pending C Macaca mulatta (Rhesus macaque) 6.1 1Present intact C Macaca nemestrina (pigtail macaque) 6.4 1 Presentintact C Theropithecus gelada (Gerlada baboon) 6.3 2 pending pending CColobus guereza kiku. (Kikuyu colobus) 6.8 2 Present pending B Alouattaseniculus (Red Howler monkey) 12.7  2 Absent n.a. B Callicebus moloch(Titi monkey) — 2 Absent n.a. B Pithecia pithecia (white-faced Saki) — 2Absent n.a. B Sanguinus fuscicollis (Saddleback tamarin) — 2 Absent n.a.B Callithrix pygmaea (Pygmy marmoset) — 2 Absent n.a. B Ateles geoffroyi(Black handed spider mky) 11.7  1 Absent n.a. A Sanguinus labiatus(Red-bellied tamarin) — 1 Absent n.a. A Lemur catta (Ring-tailed lemur)˜21.3  1 Absent n.a. A Varecia variegata ruber (lemur) — 2 Absent n.a. AOtolemur crassicaudatus (galago) 28.1 2 Absent n.a. A Nycticebus coucang(Bengal slow loris) — 2 Absent n.a.

[0138] Our inferences from these results are: a) The α-defensinprecursor of the retrocyclin gene appeared after the Old World (B) andNew World (C) monkey lineages separated. The retrocyclin gene appearedin Old World Monkeys (B) and persisted until the orangutan lineage splitfrom the lineage of Gorillas/ Chimpanzees/ Humans, some 15 million yearsago.

[0139] In a recent search of the NCBI human genome data base, twoadditional human retrocyclin genes were identified. Each gene contains asilencing mutation in its signal sequence. Two of the three retrocyclingenes encodes the same nonapeptide precursor. The third retrocyclin geneencodes a variant nonapeptide with a glycine to arginine mutation.

[0140] In the Table 4, “Hs8_(—)19639” is the NCBI identifier for theHomo sapiens chromosome 8 working draft sequence segment(Length=3,410,705 bp). The retrocyclin nonapeptide is underlined. TABLE4 Chromosomal Hs8_19639 location Translated sequence Chromosome 82,814,641 (SEQ ID NO:125) V T P A Met R T F A L L T A Met L (SEQ IDNO:124) L L V A L Stop A Q A E P L Q A R A D E A A A Q E Q P G A D D Q EMet A H A F T W H E S A A L P L S 2,813,782 S D S A R G L R C I C G R GI C R L L Stop R R F G S C A F R G T L H R I C C Chromosome 8 1,742,910(SEQ ID NO:121) V T P A Met R T F A L L T A Met L (SEQ ID NO:120) L L VA L Stop A Q A E P L Q A R A D E A A A Q E Q P G A D D Q E Met A H A F TW H E S A A L P L S 1,742,051 S D S A R G L R C I C G R R I C R L L StopR R F G S C A F R G T L H R I C C Chromosome 8 1,478,367 (SEQ ID NO:123)V T P A Met R T F A L L T A Met L (SEQ ID NO:122) L L V A L Stop A Q A EH F R Q F L Met K L Q P R S S L E Q Met I H K W L Met L Y Met A Stop K CR S S A F 1,479,224 S D S A R G L R C I C G R G I C R L L Stop R R F G SC A F R G T L H R I C C

[0141] Based on the above information, the primate ancestor of Homosapiens could have expressed three retrocyclins. The structures of thesepeptides, called Retrocyclin 1, 2 and 3 are shown in FIG. 9.

[0142] Three orangutan clones represent at least two differentretrocyclin genes. The sequences are shown in FIG. 15. The stop codonsin orangutan clone 19 are identical to those in human retrocyclin.Accordingly, clone 19 also represents an expressed pseudogene. Overall,132/143 (92.3%) of translated products (including stop codons) fromorangutan clone 19 and the human retrocyclin gene are identical. Thetranslation products of orangutan clones 20 and 21 are identical in141/143 (98.6%) sites. Both clones lack a silencing stop codon in theirsignal sequence, and should be capable of producing a functionaldemidefensin whose tandem nonapeptide elements (underlined) wouldproduce a peptide identical to human retrocyclin. The predictedtranslation products of orangutan clone 20 and human retrocyclin areidentical in 129/143 (90.2%) of positions. All three orangutan clones,#19, 20 and 21 came from the DNA of a single orangutan, It remains to bedetermined if the genes they represent are alleles, or if theretrocyclin locus has undergone duplication and additional retrocyclingenes remain to be found.

[0143] As shown in FIG. 15, this portion of the human retrocyclin geneencodes four stop codons (). The first of these occurs near the end ofthe putative signal sequence and should abort translation. The secondstop codon occurs after cysteine 3, and marks the end of the putativedemidefensin sequence. The third stop codon comes after the CCR residuesand marks the customary termination of an -defensin. The final stopcodon occurs after the FES tripeptide in a non-expressed region of thegene.

[0144] One Gorilla retrocyclin clone has been sequenced. Its translationproduct is identical to human retrocyclin in 139/143 (97.2%). Thesequence is shown in an alignment with the human sequence in FIG. 15.The silencing stop codon () is present in the signal sequence.Consequently, this clone represents an expressed pseudogene.

[0145] Note that the chimp (Pan troglodytes) and the Bonobo (Panpaniscus) genes contain the first stop codon () in the signal sequence,but both lack the retrocyclin-geherating stop codon after cysteine 3 inthe defensin-region. From these features, the chimp would appear to havesilenced an α-defensin gene. There is an additional mutation (cysteineto glycine) in the chimp's nonapeptide region (double underlined), whichwas presumably acquired after the gene had been silenced by the signalsequence mutation)

[0146] Unlike human retrocyclin, the pigtail and rhesus macaque geneslack a stop codon () in their signal sequences. Both macaque genes haveacquired a stop codon in a nontranslated portion of their gene, betweencysteines 4 and 5 of the original defensin domain.

[0147] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0148] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1 125 1 18 PRT Homo sapiens 1 Gly Ile Cys Arg Cys Ile Cys Gly Arg GlyIle Cys Arg Cys Ile Cys 1 5 10 15 Gly Arg 2 18 PRT Artificial Sequencesynthetic variant 2 Gly Ile Cys Arg Cys Ile Cys Gly Lys Gly Ile Cys ArgCys Ile Cys 1 5 10 15 Gly Arg 3 18 PRT Artificial Sequence syntheticvariant 3 Gly Ile Cys Arg Cys Tyr Cys Gly Arg Gly Ile Cys Arg Cys IleCys 1 5 10 15 Gly Arg 4 18 PRT Artificial Sequence synthetic variant 4Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys Tyr Cys 1 5 1015 Gly Arg 5 18 PRT Artificial Sequence synthetic variant 5 Gly Tyr CysArg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys Ile Cys 1 5 10 15 Gly Arg 618 PRT Artificial Sequence synthetic variant 6 Gly Ile Cys Arg Cys IleCys Gly Arg Gly Tyr Cys Arg Cys Ile Cys 1 5 10 15 Gly Arg 7 18 PRTArtificial Sequence synthetic variant 7 Gly Ile Cys Tyr Cys Ile Cys GlyArg Gly Ile Cys Arg Cys Ile Cys 1 5 10 15 Gly Arg 8 18 PRT ArtificialSequence synthetic variant 8 Gly Ile Cys Ile Cys Ile Cys Gly Tyr Gly IleCys Arg Cys Ile Cys 1 5 10 15 Gly Arg 9 18 PRT Artificial Sequencesynthetic variant 9 Gly Ile Cys Ile Cys Ile Cys Gly Arg Gly Ile Cys TyrCys Ile Cys 1 5 10 15 Gly Arg 10 18 PRT Artificial Sequence syntheticvariant 10 Arg Gly Cys Ile Cys Arg Cys Ile Gly Arg Gly Cys Ile Cys ArgCys 1 5 10 15 Ile Gly 11 496 DNA Homo sapiens CDS (124)...(304)retrocyclin 11 ggagacccgg gacagaggac tgctgtctgc cctccctctt cactctgcctaccttgagga 60 tctgtcaccc cagccatgag gaccttcgcc ctcctcactg ccatgcttctcctggtggcc 120 ctg tag gct cag gcg gag cca ctt cag gca aga gct gat gaagct gca 168 * Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu Ala Ala 15 10 gcc cag gag cag cct gga gca gat gat cag gaa atg gct cat gcc ttt 216Ala Gln Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Ala His Ala Phe 15 20 2530 aca tgg cat gaa agt gcc gct ctt ccg ctt tca gac tca gcg aga ggc 264Thr Trp His Glu Ser Ala Ala Leu Pro Leu Ser Asp Ser Ala Arg Gly 35 40 45ttg agg tgc att tgc gga aga gga att tgc cgt ttg tta t aacgtcgct t 314Leu Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Leu Leu 50 55 tgggtcctgcgcctttcgtg gtacactcca ccggatctgc tgccgctgag cttgcagaat 374 caagaaacataagctcagaa tttactttga gagttaaaag aaattcttgt tactcctgta 434 ccttgtcctccatttccttt tctcatccaa aataaatacc ttgttgcaag atttctctct 494 tt 496 12 59PRT Homo sapiens 12 Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu AlaAla Ala Gln 1 5 10 15 Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Ala HisAla Phe Thr Trp 20 25 30 His Glu Ser Ala Ala Leu Pro Leu Ser Asp Ser AlaArg Gly Leu Arg 35 40 45 Cys Ile Cys Gly Arg Gly Ile Cys Arg Leu Leu 5055 13 97 PRT Homo sapiens PEPTIDE (1)...(97) Human defensin 4 13 Met ArgIle Ile Ala Leu Leu Ala Ala Ile Leu Leu Val Ala Leu Gln 1 5 10 15 ValArg Ala Gly Pro Leu Gln Ala Arg Gly Asp Glu Ala Pro Gly Gln 20 25 30 GluGln Arg Gly Pro Glu Asp Gln Asp Ile Ser Ile Ser Phe Ala Trp 35 40 45 AspLys Ser Ser Ala Leu Gln Val Ser Gly Ser Thr Arg Gly Met Val 50 55 60 CysSer Cys Arg Leu Val Phe Cys Arg Arg Thr Glu Leu Arg Val Gly 65 70 75 80Asn Cys Leu Ile Gly Gly Val Ser Phe Thr Tyr Cys Cys Thr Arg Val 85 90 95Asp 14 500 DNA Macaca mulatta CDS (95)...(325) theta defensin 1Aprecursor 14 gacggctgct gttgctacag gagacccagg acagaggact gctgtctgcactctctcttc 60 actctgccta acttgaggat ctgtcactcc agcc atg agg acc ttc gccctc ctc 115 Met Arg Thr Phe Ala Leu Leu -20 -15 acc gcc atg ctt ctc ctggtg gcc ctg cac gct cag gca gag gca cgt 163 Thr Ala Met Leu Leu Leu ValAla Leu His Ala Gln Ala Glu Ala Arg -10 -5 1 cag gca aga gct gat gaa gctgcc gcc cag cag cag cct gga aca gat 211 Gln Ala Arg Ala Asp Glu Ala AlaAla Gln Gln Gln Pro Gly Thr Asp 5 10 15 gat cag gga atg gct cat tcc tttaca tgg cct gaa aac gcc gct ctt 259 Asp Gln Gly Met Ala His Ser Phe ThrTrp Pro Glu Asn Ala Ala Leu 20 25 30 35 cca ctt tca gag tca gcg aaa ggcttg agg tgc att tgc aca cga gga 307 Pro Leu Ser Glu Ser Ala Lys Gly LeuArg Cys Ile Cys Thr Arg Gly 40 45 50 ttc tgc cgt ttg tta taa tgtcaccttgggtcctgcgc ttttcgtggt 355 Phe Cys Arg Leu Leu * 55 tgactccacc ggatctgctgccgctgagct tccagaatca agaaaaatat gctcagaagt 415 tactttgaga gttaaaagaaattcttgcta ctgctgtacc ttctcctcag tttccttttc 475 tcatcccaaa taaataccttatcgc 500 15 76 PRT Macaca mulatta SIGNAL (1)...(20) 15 Met Arg Thr PheAla Leu Leu Thr Ala Met Leu Leu Leu Val Ala Leu -20 -15 -10 -5 His AlaGln Ala Glu Ala Arg Gln Ala Arg Ala Asp Glu Ala Ala Ala 1 5 10 Gln GlnGln Pro Gly Thr Asp Asp Gln Gly Met Ala His Ser Phe Thr 15 20 25 Trp ProGlu Asn Ala Ala Leu Pro Leu Ser Glu Ser Ala Lys Gly Leu 30 35 40 Arg CysIle Cys Thr Arg Gly Phe Cys Arg Leu Leu 45 50 55 16 495 DNA Macacamulatta CDS (90)...(320) theta defensin 1b precursor 16 gaccgctgctcttgctacag gagacccggg acagaggact gctgtctgcc ctctctcttc 60 actctgcctaacttgaggat ctgccagcc atg agg acc ttc gcc ctc ctc acc 113 Met Arg Thr PheAla Leu Leu Thr -20 -15 gcc atg ctt ctc ctg gtg gcc ctg cac gct cag gcagag gca cgt cag 161 Ala Met Leu Leu Leu Val Ala Leu His Ala Gln Ala GluAla Arg Gln -10 -5 1 gca aga gct gat gaa gct gcc gcc cag cag cag cct ggagca gat gat 209 Ala Arg Ala Asp Glu Ala Ala Ala Gln Gln Gln Pro Gly AlaAsp Asp 5 10 15 20 cag gga atg gct cat tcc ttt aca cgg cct gaa aac gccgct ctt ccg 257 Gln Gly Met Ala His Ser Phe Thr Arg Pro Glu Asn Ala AlaLeu Pro 25 30 35 ctt tca gag tca gcg aga ggc ttg agg tgc ctt tgc aga cgagga gtt 305 Leu Ser Glu Ser Ala Arg Gly Leu Arg Cys Leu Cys Arg Arg GlyVal 40 45 50 tgc caa ctg tta taa aggcgtttgg ggtcctgcgc ttttcgtggttgactctgcc 360 Cys Gln Leu Leu * 55 ggatctgctg ccgctgagct tccagaatcaagaaaaatac gctcagaagt tactttgaga 420 gttgaaagaa attcctgtta ctcctgtaccttgtcctcaa tttccttttc tcatcccaaa 480 taaatacctt ctcgc 495 17 76 PRTMacaca mulatta SIGNAL (1)...(20) 17 Met Arg Thr Phe Ala Leu Leu Thr AlaMet Leu Leu Leu Val Ala Leu -20 -15 -10 -5 His Ala Gln Ala Glu Ala ArgGln Ala Arg Ala Asp Glu Ala Ala Ala 1 5 10 Gln Gln Gln Pro Gly Ala AspAsp Gln Gly Met Ala His Ser Phe Thr 15 20 25 Arg Pro Glu Asn Ala Ala LeuPro Leu Ser Glu Ser Ala Arg Gly Leu 30 35 40 Arg Cys Leu Cys Arg Arg GlyVal Cys Gln Leu Leu 45 50 55 18 54 DNA Artificial Sequence syntheticsequence 18 aggtgcattt gcggaagagg aatttgcagg tgcatttgcg gaagaggaat ttgc54 19 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 19 Arg Cys Ile Cys Gly Arg Gly Ile Cys 1 5 20 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 20 Arg Cys Leu Cys Gly Arg Gly Ile Cys 1 5 21 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 21Arg Cys Ile Cys Arg Arg Gly Ile Cys 1 5 22 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 22 Arg CysIle Cys Thr Arg Gly Ile Cys 1 5 23 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 23 Arg Cys Ile Cys ValArg Gly Ile Cys 1 5 24 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 24 Arg Cys Ile Cys Gly LeuGly Ile Cys 1 5 25 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 25 Arg Cys Ile Cys Gly Arg Gly Val Cys 15 26 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 26 Arg Cys Ile Cys Gly Arg Gly Phe Cys 1 5 27 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 27 Arg Cys Leu Cys Arg Arg Gly Val Cys 1 5 28 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 28Arg Cys Leu Cys Thr Arg Gly Ile Cys 1 5 29 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 29 Arg CysLeu Cys Val Arg Gly Ile Cys 1 5 30 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 30 Arg Cys Leu Cys GlyLeu Gly Val Cys 1 5 31 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 31 Arg Cys Leu Cys Gly ArgGly Val Cys 1 5 32 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 32 Arg Cys Leu Cys Gly Arg Gly Phe Cys 15 33 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 33 Arg Cys Ile Cys Arg Arg Gly Val Cys 1 5 34 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 34 Arg Cys Ile Cys Arg Arg Gly Phe Cys 1 5 35 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 35Arg Cys Ile Cys Thr Arg Gly Val Cys 1 5 36 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 36 Arg CysIle Cys Thr Arg Gly Phe Cys 1 5 37 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 37 Arg Cys Ile Cys ThrLeu Gly Ile Cys 1 5 38 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 38 Arg Cys Ile Cys Val LeuGly Phe Cys 1 5 39 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 39 Arg Cys Ile Cys Arg Leu Gly Ile Cys 15 40 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 40 Arg Cys Ile Cys Val Arg Gly Val Cys 1 5 41 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 41 Arg Cys Ile Cys Gly Arg Gly Phe Cys 1 5 42 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 42Arg Cys Ile Cys Gly Leu Gly Phe Cys 1 5 43 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 43 Arg CysIle Cys Gly Leu Gly Val Cys 1 5 44 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 44 Arg Cys Leu Cys ArgLeu Gly Ile Cys 1 5 45 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 45 Arg Cys Leu Cys Arg ArgGly Val Cys 1 5 46 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 46 Arg Cys Leu Cys Arg Arg Gly Phe Cys 15 47 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 47 Arg Cys Leu Cys Thr Leu Gly Ile Cys 1 5 48 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 48 Arg Cys Leu Cys Thr Arg Gly Val Cys 1 5 49 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 49Arg Cys Leu Cys Thr Arg Gly Phe Cys 1 5 50 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 50 Arg CysLeu Cys Val Leu Gly Ile Cys 1 5 51 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 51 Arg Cys Leu Cys ValArg Gly Val Cys 1 5 52 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 52 Arg Cys Ile Cys Gly ArgGly Ile Cys 1 5 53 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 53 Arg Cys Ile Cys Arg Leu Gly Val Cys 15 54 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 54 Arg Cys Ile Cys Arg Leu Gly Phe Cys 1 5 55 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 55 Arg Cys Ile Cys Thr Leu Gly Val Cys 1 5 56 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 56Arg Cys Ile Cys Thr Leu Gly Phe Cys 1 5 57 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 57 Arg CysIle Cys Val Leu Gly Val Cys 1 5 58 9 PRT Artificial Sequence generatedby replacement of variants in consensus sequence 58 Arg Cys Ile Cys ValLeu Gly Phe Cys 1 5 59 9 PRT Artificial Sequence generated byreplacement of variants in consensus sequence 59 Arg Cys Leu Cys Gly LeuGly Val Cys 1 5 60 9 PRT Artificial Sequence generated by replacement ofvariants in consensus sequence 60 Arg Cys Leu Cys Gly Leu Gly Ile Cys 15 61 9 PRT Artificial Sequence generated by replacement of variants inconsensus sequence 61 Arg Cys Leu Cys Thr Leu Gly Val Cys 1 5 62 9 PRTArtificial Sequence generated by replacement of variants in consensussequence 62 Arg Cys Leu Cys Thr Leu Gly Ile Cys 1 5 63 9 PRT ArtificialSequence generated by replacement of variants in consensus sequence 63Arg Cys Leu Cys Val Leu Gly Val Cys 1 5 64 9 PRT Artificial Sequencegenerated by replacement of variants in consensus sequence 64 Arg CysLeu Cys Val Leu Gly Ile Cys 1 5 65 140 PRT Homo sapiens 65 Val Thr ProAla Met Arg Thr Phe Ala Leu Leu Thr Ala Met Leu Leu 1 5 10 15 Leu ValAla Leu Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu 20 25 30 Ala AlaAla Gln Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Ala His 35 40 45 Ala PheThr Trp His Glu Ser Ala Ala Leu Pro Leu Ser Asp Ser Ala 50 55 60 Arg GlyLeu Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Leu Leu Arg 65 70 75 80 ArgPhe Gly Ser Cys Ala Phe Arg Gly Thr Leu His Arg Ile Cys Cys 85 90 95 ArgAla Cys Arg Ile Lys Lys His Lys Leu Arg Ile Tyr Phe Glu Ser 100 105 110Lys Lys Phe Leu Leu Leu Leu Tyr Leu Val Leu His Phe Leu Phe Ser 115 120125 Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Ser Leu 130 135 140 66 140PRT Orangutan 66 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Leu Ala Ala MetLeu Leu 1 5 10 15 Leu Val Ala Leu Ala Glu Ala Glu Pro Leu Gln Ala ArgAla Asp Glu 20 25 30 Thr Ala Ala Gln Glu Gln Pro Gly Ala Asp Asp Gln GluMet Ala His 35 40 45 Ala Phe Thr Trp Asp Glu Ser Ala Thr Leu Pro Leu SerAsp Ser Ala 50 55 60 Arg Gly Leu Arg Cys Ile Cys Arg Arg Gly Val Cys ArgPhe Leu Arg 65 70 75 80 His Leu Gly Ser Cys Ala Phe Arg Gly Thr Leu HisArg Ile Cys Cys 85 90 95 Arg Ala Cys Arg Ile Lys Lys Asn Lys Leu Arg IleTyr Phe Glu Ser 100 105 110 Lys Lys Phe Val Phe Leu Leu Tyr Leu Ala LeuHis Phe Leu Phe Ser 115 120 125 Ser Lys Ile Asn Thr Leu Leu Gln Asp PheCys Leu 130 135 140 67 141 PRT Orangutan 67 Val Thr Pro Ala Met Arg ThrPhe Thr Val Leu Ala Ala Met Leu Leu 1 5 10 15 Val Val Ala Leu Gln AlaGln Ala Glu Pro Leu Arg Ala Arg Ala Asp 20 25 30 Glu Thr Ala Ala Gln GluGln Pro Gly Ala Asp Asp Gln Glu Met Ala 35 40 45 His Ala Phe Thr Trp AspGlu Ser Ala Ala Leu Pro Leu Ser Asp Ser 50 55 60 Ala Arg Gly Leu Arg CysIle Cys Arg Arg Gly Val Cys Arg Phe Leu 65 70 75 80 Arg His Leu Gly SerCys Ala Phe Arg Gly Thr Leu His Arg Ile Cys 85 90 95 Cys Arg Ala Cys ArgIle Lys Lys Asn Lys Leu Arg Ile Tyr Phe Glu 100 105 110 Ser Lys Lys PheVal Phe Leu Leu Tyr Leu Ala Leu His Phe Leu Phe 115 120 125 Ser Ser LysIle Asn Thr Leu Leu Gln Asp Phe Cys Leu 130 135 140 68 141 PRT Orangutan68 Val Thr Pro Ala Met Arg Thr Phe Thr Val Leu Ala Ala Met Leu Leu 1 510 15 Val Val Ala Leu Gln Ala Gln Ala Glu Pro Leu Arg Ala Arg Ala Asp 2025 30 Glu Thr Ala Ala Gln Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Ala 3540 45 His Ala Phe Thr Trp Asp Glu Ser Ala Ala Leu Pro Leu Ser Asp Ser 5055 60 Ala Arg Gly Leu Arg Cys Ile Cys Arg Arg Gly Val Cys Arg Leu Leu 6570 75 80 Arg His Phe Gly Ser Cys Ala Phe Arg Gly Thr Leu His Arg Ile Cys85 90 95 Cys Arg Ala Cys Arg Ile Lys Lys Asn Lys Leu Arg Ile Tyr Phe Glu100 105 110 Ser Lys Lys Phe Leu Phe Leu Leu Tyr Leu Ala Leu His Phe LeuPhe 115 120 125 Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Cys Leu 130135 140 69 140 PRT Gorilla 69 Val Thr Pro Ala Met Arg Thr Phe Ala LeuLeu Thr Ala Met Leu Leu 1 5 10 15 Leu Val Asp Leu Ala Gln Ala Glu ProLeu Gln Ala Arg Ala Asp Glu 20 25 30 Ala Ala Ala Gln Glu Gln Pro Gly AlaAsp Asp Gln Glu Met Ala His 35 40 45 Ala Phe Thr Trp Asp Glu Ser Ala AlaLeu Pro Leu Ser Asp Ser Ala 50 55 60 Arg Gly Leu Arg Cys Ile Cys Gly ArgGly Ile Cys Arg Leu Leu Arg 65 70 75 80 Arg Phe Gly Ser Cys Ala Phe ArgGly Thr Leu His Arg Ile Cys Cys 85 90 95 Arg Ala Cys Arg Ile Lys Lys AsnLys Leu Arg Ile Tyr Phe Glu Thr 100 105 110 Lys Lys Phe Leu Leu Leu LeuTyr Leu Val Leu His Phe Leu Phe Ser 115 120 125 Ser Lys Ile Asn Thr LeuLeu Gln Asp Phe Cys Leu 130 135 140 70 141 PRT Champanzee 70 Val Thr ProAla Met Arg Thr Phe Ala Leu Leu Thr Ala Met Leu Leu 1 5 10 15 Leu ValAla Leu Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu 20 25 30 Ala AlaAla Gln Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Ala His 35 40 45 Ala PheThr Trp Asp Glu Ser Ala Ala Leu Pro Leu Ser Asp Ser Ala 50 55 60 Arg GlyLeu Arg Cys Ile Gly Gly Arg Gly Ile Cys Gly Leu Leu Gln 65 70 75 80 ArgArg Phe Gly Ser Cys Ala Phe Arg Gly Thr Leu His Arg Ile Cys 85 90 95 CysArg Ala Cys Arg Ile Lys Lys Asn Lys Leu Arg Ile Tyr Ser Glu 100 105 110Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu Val Leu His Phe Leu Phe 115 120125 Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Ser Leu 130 135 140 71141 PRT Chimpanzee 71 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Leu ThrAla Met Leu Leu 1 5 10 15 Leu Val Ala Leu Ala Gln Ala Glu Pro Leu GlnAla Arg Ala Asp Glu 20 25 30 Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp AspGln Glu Met Ala His 35 40 45 Ala Phe Thr Trp Asp Glu Ser Ala Ala Leu ProLeu Ser Asp Ser Ala 50 55 60 Arg Gly Leu Arg Cys Ile Gly Gly Arg Gly IleCys Gly Leu Leu Gln 65 70 75 80 Arg Arg Val Gly Ser Cys Ala Phe Arg GlyThr Leu His Arg Ile Cys 85 90 95 Cys Arg Ala Cys Arg Ile Lys Lys Asn LysLeu Arg Ile Tyr Ser Glu 100 105 110 Ser Lys Lys Phe Leu Leu Leu Leu TyrLeu Val Leu His Phe Leu Phe 115 120 125 Ser Ser Lys Ile Asn Thr Ser LeuGln Asp Phe Ser Leu 130 135 140 72 141 PRT Rhesus monkey VARIANT 113 Xaa= Any Amino Acid 72 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Leu Thr AlaMet Leu Leu 1 5 10 15 Leu Val Ala Leu His Ala Gln Ala Glu Ala Arg GlnAla Arg Ala Asp 20 25 30 Glu Ala Ala Ala Gln Gln Gln Pro Gly Ala Asp AspGln Gly Met Ala 35 40 45 His Ser Phe Thr Arg Pro Glu Asn Ala Ala Leu ProLeu Ser Glu Ser 50 55 60 Ala Arg Gly Leu Arg Cys Leu Cys Arg Arg Gly ValCys Gln Leu Leu 65 70 75 80 Arg Arg Leu Gly Ser Cys Ala Phe Arg Gly LeuCys Arg Ile Cys Cys 85 90 95 Arg Ala Ser Arg Ile Lys Lys Asn Thr Leu ArgSer Tyr Phe Glu Ser 100 105 110 Xaa Lys Lys Phe Leu Leu Leu Leu Tyr LeuVal Leu Asn Phe Leu Phe 115 120 125 Ser Ser Gln Ile Asn Thr Phe Ser GlnAsp Phe Cys Leu 130 135 140 73 141 PRT Pig-tailed macaque VARIANT 113Xaa = Any Amino Acid 73 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Leu ThrAla Met Leu Leu 1 5 10 15 Leu Val Ala Leu His Ala Gln Ala Glu Ala ArgGln Ala Arg Ala Asp 20 25 30 Glu Ala Ala Ala Gln Gln Gln Pro Gly Ala AspAsp Gln Gly Met Ala 35 40 45 His Ser Phe Thr Arg Pro Glu Asn Ala Ala LeuPro Leu Ser Glu Ser 50 55 60 Ala Arg Gly Leu Arg Cys Ile Cys Arg Arg GlyVal Cys Gln Leu Leu 65 70 75 80 Arg Arg Leu Gly Ser Cys Ala Phe Arg GlyLeu Cys Arg Ile Cys Cys 85 90 95 Arg Ala Ser Arg Ile Lys Lys Asn Thr LeuArg Ser Tyr Phe Glu Ser 100 105 110 Xaa Lys Lys Phe Leu Leu Leu Leu TyrLeu Val Leu Asn Phe Leu Phe 115 120 125 Ser Ser Gln Ile Asn Thr Phe SerGln Asp Phe Cys Leu 130 135 140 74 9 PRT Artificial Sequence expansionof variant residues. 74 Arg Cys Ile Cys Gly Arg Arg Ile Cys 1 5 75 9 PRTArtificial Sequence expansion of variant residues. 75 Arg Cys Leu CysGly Arg Arg Ile Cys 1 5 76 9 PRT Artificial Sequence expansion ofvariant residues. 76 Arg Cys Ile Cys Arg Arg Arg Ile Cys 1 5 77 9 PRTArtificial Sequence expansion of variant residues. 77 Arg Cys Ile CysThr Arg Arg Ile Cys 1 5 78 9 PRT Artificial Sequence expansion ofvariant residues. 78 Arg Cys Ile Cys Val Arg Arg Ile Cys 1 5 79 9 PRTArtificial Sequence expansion of variant residues. 79 Arg Cys Ile CysGly Leu Arg Ile Cys 1 5 80 9 PRT Artificial Sequence expansion ofvariant residues. 80 Arg Cys Ile Cys Gly Arg Arg Val Cys 1 5 81 9 PRTArtificial Sequence expansion of variant residues. 81 Arg Cys Ile CysGly Arg Arg Phe Cys 1 5 82 9 PRT Artificial Sequence expansion ofvariant residues. 82 Arg Cys Leu Cys Arg Arg Arg Val Cys 1 5 83 9 PRTArtificial Sequence expansion of variant residues. 83 Arg Cys Leu CysThr Arg Arg Ile Cys 1 5 84 9 PRT Artificial Sequence expansion ofvariant residues. 84 Arg Cys Leu Cys Val Arg Arg Ile Cys 1 5 85 9 PRTArtificial Sequence expansion of variant residues. 85 Arg Cys Leu CysGly Leu Arg Val Cys 1 5 86 9 PRT Artificial Sequence expansion ofvariant residues. 86 Arg Cys Leu Cys Gly Arg Arg Val Cys 1 5 87 9 PRTArtificial Sequence expansion of variant residues. 87 Arg Cys Leu CysGly Arg Arg Phe Cys 1 5 88 9 PRT Artificial Sequence expansion ofvariant residues. 88 Arg Cys Ile Cys Arg Arg Arg Val Cys 1 5 89 9 PRTArtificial Sequence expansion of variant residues. 89 Arg Cys Ile CysArg Arg Arg Phe Cys 1 5 90 9 PRT Artificial Sequence expansion ofvariant residues. 90 Arg Cys Ile Cys Thr Arg Arg Val Cys 1 5 91 9 PRTArtificial Sequence expansion of variant residues. 91 Arg Cys Ile CysThr Arg Arg Phe Cys 1 5 92 9 PRT Artificial Sequence expansion ofvariant residues. 92 Arg Cys Ile Cys Thr Leu Arg Ile Cys 1 5 93 9 PRTArtificial Sequence expansion of variant residues. 93 Arg Cys Ile CysVal Leu Arg Phe Cys 1 5 94 9 PRT Artificial Sequence expansion ofvariant residues. 94 Arg Cys Ile Cys Arg Leu Arg Ile Cys 1 5 95 9 PRTArtificial Sequence expansion of variant residues. 95 Arg Cys Ile CysVal Arg Arg Val Cys 1 5 96 9 PRT Artificial Sequence expansion ofvariant residues. 96 Arg Cys Ile Cys Gly Arg Arg Phe Cys 1 5 97 9 PRTArtificial Sequence expansion of variant residues. 97 Arg Cys Ile CysGly Leu Arg Phe Cys 1 5 98 9 PRT Artificial Sequence expansion ofvariant residues. 98 Arg Cys Ile Cys Gly Leu Arg Val Cys 1 5 99 9 PRTArtificial Sequence expansion of variant residues. 99 Arg Cys Leu CysArg Leu Arg Ile Cys 1 5 100 9 PRT Artificial Sequence expansion ofvariant residues. 100 Arg Cys Leu Cys Arg Arg Arg Val Cys 1 5 101 9 PRTArtificial Sequence expansion of variant residues. 101 Arg Cys Leu CysArg Arg Arg Phe Cys 1 5 102 9 PRT Artificial Sequence expansion ofvariant residues. 102 Arg Cys Leu Cys Thr Leu Arg Ile Cys 1 5 103 9 PRTArtificial Sequence expansion of variant residues. 103 Arg Cys Leu CysGly Arg Arg Val Cys 1 5 104 9 PRT Artificial Sequence expansion ofvariant residues. 104 Arg Cys Leu Cys Thr Arg Arg Phe Cys 1 5 105 9 PRTArtificial Sequence expansion of variant residues. 105 Arg Cys Leu CysVal Leu Arg Ile Cys 1 5 106 9 PRT Artificial Sequence expansion ofvariant residues. 106 Arg Cys Leu Cys Val Arg Arg Val Cys 1 5 107 9 PRTArtificial Sequence expansion of variant residues. 107 Arg Cys Ile CysGly Arg Arg Ile Cys 1 5 108 9 PRT Artificial Sequence expansion ofvariant residues. 108 Arg Cys Ile Cys Arg Leu Arg Val Cys 1 5 109 9 PRTArtificial Sequence expansion of variant residues. 109 Arg Cys Ile CysArg Leu Arg Phe Cys 1 5 110 9 PRT Artificial Sequence expansion ofvariant residues. 110 Arg Cys Ile Cys Thr Leu Arg Val Cys 1 5 111 9 PRTArtificial Sequence expansion of variant residues. 111 Arg Cys Ile CysThr Leu Arg Phe Cys 1 5 112 9 PRT Artificial Sequence expansion ofvariant residues. 112 Arg Cys Ile Cys Val Leu Arg Val Cys 1 5 113 9 PRTArtificial Sequence expansion of variant residues. 113 Arg Cys Ile CysVal Leu Arg Phe Cys 1 5 114 9 PRT Artificial Sequence expansion ofvariant residues. 114 Arg Cys Leu Cys Gly Leu Arg Val Cys 1 5 115 9 PRTArtificial Sequence expansion of variant residues. 115 Arg Cys Leu CysGly Leu Arg Ile Cys 1 5 116 9 PRT Artificial Sequence expansion ofvariant residues. 116 Arg Cys Leu Cys Thr Leu Arg Val Cys 1 5 117 9 PRTArtificial Sequence expansion of variant residues. 117 Arg Cys Leu CysThr Leu Arg Ile Cys 1 5 118 9 PRT Artificial Sequence expansion ofvariant residues. 118 Arg Cys Leu Cys Val Leu Arg Val Cys 1 5 119 9 PRTArtificial Sequence expansion of variant residues. 119 Arg Cys Leu CysVal Leu Arg Ile Cys 1 5 120 437 DNA Homo sapiens 120 gtcaccccagccatgaggac cttcgccctc ctcactgcca tgcttctcct ggtggccctg 60 taggctcaggcggagccact tcaggcaaga gctgatgaag ctgcagccca ggagcagcct 120 ggagcagatgatcaggaaat ggctcatgcc tttacatggc atgaaagtgc cgctcttccg 180 ttttcagtcagactcagcga gaggcttgag gtgcatttgc ggaagaggaa tttgccgttt 240 gttataacgtcgctttgggt cctgcgcctt tcgtggtaca ctccaccggg tctgctgccg 300 ctgaacttgcagaatcaaga aaaataagct cagaatttac tttgagagtt aaaagaaatt 360 cttgttactcctgtaccttg tcctccattt ccttttctca tccaaaataa ataccttgtt 420 gcaagatttctctcttt 437 121 118 PRT Homo sapiens 121 Val Thr Pro Ala Met Arg Thr PheAla Leu Leu Thr Ala Met Leu Leu 1 5 10 15 Leu Val Ala Leu Ala Gln AlaGlu Pro Leu Gln Ala Arg Ala Asp Glu 20 25 30 Ala Ala Ala Gln Glu Gln SerAsp Ser Ala Arg Gly Leu Arg Cys Ile 35 40 45 Cys Gly Arg Gly Ile Cys ArgLeu Leu Arg Arg Phe Gly Ser Cys Ala 50 55 60 Phe Arg Gly Thr Leu His ArgVal Cys Cys Arg Thr Cys Arg Ile Lys 65 70 75 80 Lys Asn Lys Leu Arg IleTyr Phe Glu Ser Lys Lys Phe Leu Leu Leu 85 90 95 Leu Tyr Leu Val Leu HisPhe Leu Phe Ser Ser Lys Ile Asn Thr Leu 100 105 110 Leu Gln Asp Phe SerLeu 115 122 437 DNA Homo sapiens 122 gtcaccccag ccatgaggac ctttgccctcctcactgcca tgcttctcct ggtggccctg 60 taggctcagg cagagccact tcaggcaagagctgatgaag ctgcagccca ggagcagcct 120 ggagcagatg atcaggaaat ggctcatgcctttacatggc atgaaagtgc cgctcttccg 180 ctttcagtca gactcagcga gaggcttgaggtgcatttgc ggaagaagaa tttgccgttt 240 gttataacgt cgctttgggt cctgcgcctttcgtggtaca ctccaccgga tctgctgccg 300 ctgagcttgc agaatcaaga aacataagctcagaatttac tttgagagtt aaaagaaatt 360 cttgttactc ctgtaccttg tcctccatttccttttctca tccaaaataa ataccttgtt 420 gcaagatttc tctcttt 437 123 141 PRTHomo sapiens 123 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Leu Thr Ala MetLeu Leu 1 5 10 15 Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gln Ala ArgAla Asp Glu 20 25 30 Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp Asp Gln GluMet Ala His 35 40 45 Ala Phe Thr Trp His Glu Ser Ala Ala Leu Pro Leu SerSer Asp Ser 50 55 60 Ala Arg Gly Leu Arg Cys Ile Cys Gly Arg Arg Ile CysArg Leu Leu 65 70 75 80 Arg Arg Phe Gly Ser Cys Ala Phe Arg Gly Thr LeuHis Arg Ile Cys 85 90 95 Cys Arg Ala Cys Arg Ile Lys Lys His Lys Leu ArgIle Tyr Phe Glu 100 105 110 Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu ValLeu His Phe Leu Phe 115 120 125 Ser Ser Lys Ile Asn Thr Leu Leu Gln AspPhe Ser Leu 130 135 140 124 437 DNA Homo sapiens 124 gtcaccccagccatgaggac ctttgccctc ctcactgcca tgcttctcct ggtggccctg 60 taggctcaggcagagccact tcaggcaaga gctgatgaag ctgcagccca ggagcagcct 120 ggagcagatgatcaggaaat ggctcatgcc tttacatggc atgaaagtgc cgctcttccg 180 ctttcagtcagactcagcga gaggcttgag gtgcatttgc ggaagaggaa tttgccgttt 240 gttataacgtcgctttgggt cctgcgcctt tcgtggtaca ctccaccgga tctgctgccg 300 ctgagcttgcagaatcaaga aacataagct cagaatttac tttgagagtt aaaagaaatt 360 cttgttactcctgtaccttg tcctccattt ccttttctca tccaaaataa ataccttgtt 420 gcaagatttctctcttt 437 125 141 PRT Homo sapiens 125 Val Thr Pro Ala Met Arg Thr PheAla Leu Leu Thr Ala Met Leu Leu 1 5 10 15 Leu Val Ala Leu Ala Gln AlaGlu Pro Leu Gln Ala Arg Ala Asp Glu 20 25 30 Ala Ala Ala Gln Glu Gln ProGly Ala Asp Asp Gln Glu Met Ala His 35 40 45 Ala Phe Thr Trp His Glu SerAla Ala Leu Pro Leu Ser Ser Asp Ser 50 55 60 Ala Arg Gly Leu Arg Cys IleCys Gly Arg Gly Ile Cys Arg Leu Leu 65 70 75 80 Arg Arg Phe Gly Ser CysAla Phe Arg Gly Thr Leu His Arg Ile Cys 85 90 95 Cys Arg Ala Cys Arg IleLys Lys His Lys Leu Arg Ile Tyr Phe Glu 100 105 110 Ser Lys Lys Phe LeuLeu Leu Leu Tyr Leu Val Leu His Phe Leu Phe 115 120 125 Ser Ser Lys IleAsn Thr Leu Leu Gln Asp Phe Ser Leu 130 135 140

What is claimed is:
 1. An isolated retrocyclin peptide.
 2. The isolatedretrocyclin peptide according to claim 1, wherein said peptide isencoded by one or more primate genetic sequences.
 3. The isolatedretrocyclin peptide according to claim 2, wherein said peptide isencoded by one or more human genetic sequences.
 4. The isolatedretrocyclin peptide according to claim 1, wherein said peptide islinear.
 5. The isolated retrocyclin peptide according to claim 1,wherein said peptide is circular.
 6. The isolated retrocyclin accordingto claim 1, wherein said peptide is two linked nonapeptides, whereineach nonapeptide sequence is independently selected from the groupconsisting of SEQ ID NO: 19 to SEQ ID NO: 64; and SEQ ID NO: 74 to SEQID NO:
 119. 7. A cyclic polypeptide comprising the amino acid sequence:X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈ whereinX1 and X18 are linked through a peptide bond, disulfide crosslinks areformed between at least one of: X₃ and X₁₆; X₅ and X₁₄, and X₇ and X₁₂with the proviso that when such a crosslink is present, the crosslinkedamino acids are both cysteines; at least about three of amino acids X₁to X₁₈ are arginine or lysine; X₂, X₅, X₁₁, X₁₅ are aliphatic aminoacids; and X₁, X₁₀ and X₁₇ are glycine or alanine.
 8. The cyclicpolypeptide according to claim 7, wherein three pairs of cysteines arecrosslinked.
 9. The cyclic polypeptide of claim 7, wherein saidpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:1-10.
 10. The cyclic polypeptide of claim 7, wherein said polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 5 or 8.11. The cyclic polypeptide of claim 1, and a pharmaceutically acceptableexcipient.
 12. An isolated nucleic acid encoding a primate retrocyclinor a peptide set forth in SEQ ID NO: 1-10.
 13. An isolated nucleic acidaccording to claim 12, wherein said retrocyclin is human.
 14. Theisolated nucleic acid according to claim 13, wherein said nucleic acidcomprises the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 119, SEQID NO: 121, or SEQ ID NO:
 123. 15. A method for preventing retroviralinfection in a cell, the method comprising: administering an effectivedose of a circular minidefensin or retrocyclin to said cell. :
 16. Themethod according to claim 14, wherein said cell is of a type that issusceptible to bacterial or viral infection.
 17. A method for killingmicrobial organisms, the method comprising: administering an effectivedose of retrocyclin to said microbial organisms.
 18. A method foradministering retrocyclin as a therapeutic agent to a patient with anestablished microbial or viral infection.
 19. A method for administeringretrocyclin as a prophylactic agent to prevent a microbial or viralinfection in a patient at risk of developing such infection.